SEPARATION OF SUBCELLULAR ORGANELLES BY DIFFIERENTIAL AND DENSITY G W I E N T CENTRIFUGATION*

DARREL E. GOLL, RONALD B . YOUNG, AND MARVIN H . STROMER Iowa S ta t e University

INTRODUCTION

The centrifuge i s now f i rmly established as an indispensable t o o l Centrifugation may be used i n v i r t u a l l y a l l phases of modern biology.

e i the r : (1) t o separate a mixture of d i f f e ren t substances i n suspension or solut ion and t o i s o l a t e these substances i n t o suspensions or solut ions containing only one kind of pa r t i c l e ; o r (2 ) t o character ize the s i ze , shape and densi ty of the p a r t i c l e s after they have been separated i n t o pur i f ied solut ions or suspensions . Consequently, centr i fugat ion can be used i n both pur i f ica t ion and character izat ion of a wide range of b io logica l substances. centrifuge invaluable and very widely used i n c e l l u l a r and molecular biology. Because of i t s p rac t i ca l ly universal appl icat ion, an exorbi tant ly lengthy discussion would be required t o review a l l b io logica l uses of t he centr i fuge. Consequently, t h i s account w i l l be l imited t o a discussion of only t h e f i rs t of t he two general appl icat ions of the centrifuge; namely, use of t he centrifuge t o separate mixtures of d i f f e ren t p a r t i c l e s i n t o suspensions o r solut ions of pur i f ied p a r t i c l e s . The second general appl icat ion of the centrifuge, use of centr i fugat ion t o character ize s i z e and shape of pa r t i c l e s , w i l l not be discussed e x p l i c i t l y i n t h i s review. Whenever possible, separations involving muscle t i s s u e or subcel lular organelles from muscle t i s s u e w i l l be used t o i l l u s t r a t e t h e usef'ulness of t he centrifuge i n muscle biology and meat science.

This un iversa l i ty of appl icat ion mabs the

The mixtures of d i f f e ren t substances that are commonly fract ionated by centr i fugat ion may contain e i t h e r whole i n t a c t c e l l s o r subcel lular components of c e l l s released by rupture of t he outer c e l l membrane. For example, d i f f e ren t kinds of c e l l s may be resolved in to homogeneous c e l l populations by centr i fugat ion (Boone e t a 1 ., 1968) centr i fugat ion is used t o purify a given subcel lular component f romthe e n t i r e m i x t u r e of subcel lular components released upon rupture of the c e l l membrane. I n muscle t i s sue , centr i fugat ion has not ye t been widely used on the first of these two kinds of samples, i.e., on suspensions of

More frequently,

* Journal Paper No. 5-7956 of the Iowa Agriculture and Home Economics Experimnt S ta t ion , Ames, Projects 1.795 and 1796. s tud ies on muscle polysomes reported i n t h i s review were supported i n p a r t by g ran t s from t h e National I n s t i t u t e s of Health (AM U654), t he Muscular Dystrophy Associations of America, t h e $merican Heart Association (Grant No. 71-679), and the Iowa Heart Association (Grants No. 72-G-15 and 734-20). Presented a t t h e 27th Annual Reciprocal Meat Conference of t he American Meat Science Association, 1974.

The o r i g i n a l

i n t a c t muscle c e l l s . could be used advantageausly t o purify whole muscle c e l l s from muscle c e l l cul tures grown i n v i t r o . Fibroblasts normally contaminate cul tures of muscle c e l l s , sometimes const i tut ing up t o 3% of t o t a l c e l l s i n such cul tures , and f ib rob la s t s are very d i f f i c u l t t o separate completely from cultured muscle c e l l s by ordinary procedures. from muscle c e l l s i n several ways t h a t may cause hydrodynamic differences, it seems probable t h a t centr i fugat ion under the proper conditions would separate muscle c e l l s and f ibroblas t s , although t o the best of our know- ledge, such centr i fugal resolut ion has not yet been ser iously attempted. Even though centr i fugat ion has not been widely used f o r separation and pu r i f i ca t ion of i n t a c t muscle c e l l s , it has been used t o purify a number of other c e l l types (Boone e t a l . , 1968). For example, centr i fugat ion i n densi ty gradients has been used t o separate malignant from normal or nonmalignant c e l l s (Pretlow, 1971; Pretlow and Boone, 1968; 1970), t o separate mest cells i n d i f f e ren t stages of maturational d i f f e ren t i a t ion (Pretlow and Cassady, 1970), t o purify human leukemia c e l l s (Abeloff e t -- al. , 1970; Boone numerous similar separations (Miller and Cudkowicz, 1971). procedures f o r pur i f ica t ion of c e l l s by centr i fugat ion have been developed only recently, and it i s l i k e l y that use of the centrifuge t o prepare suspensions of homogeneous c e l l s w i l l increase i n the fu ture .

It seems l ike ly , however, t h a t centr i fugat ion

Because f ib rob la s t s d i f f e r

G., 1968; Pretlow and Boone, l969), and f o r Rotors and

FRACTIONATION OF SUBCELLULAR ORGANELLES BY DIFFERENTIAL CENTRIFUGATION

Centrifugation is probably most frequently used i n biology t o f rac t iona te the mixture OP subcel lular components produced by rupture of the outer c e l l membrane in to purified preparations containing only one kind of subcel lular pa r t i c l e , and the reminder of t h i s discussion w i l l be concerned spec i f i ca l ly with t h i s use of centr i fugat ion. It should be c l ea r ly specified, however, t h a t the pr inc ip les used t o separate subcel lular organelles by centrif'ugation a re a l so d i r e c t l y applicable t o cent r i fuga l separation of i n t a c t c e l l s , and t h a t t he only differences between cent r i fuga l separation of c e l l s and subcel lular organelles a r e the procedural protocols, such as centrifuge speed and suspending solut ion used, associated with the two d i f f e ren t kinds of sample mater ia l . A s shown i n Figures 1 and 2, s k e l e t a l muscle c e l l s a r e composed of a t l e a s t nine d i f f e ren t kinds of subcel lular components t h a t can be ident i f ied and s t r u c t u r a l l y characterized w i t h the e lectron microscope. These nine components include the nucleus, mitochondria, the sarcolemma or outer c e l l membrane, myofibrils, which a r e the con t r ac t i l e elements of muscle, T- o r transverse-tubules, the sarco- plasmic r e t i c u l a r membranes, glycogen granules, r ibosmes, and the sarcoplasm, which is not s t r u c t u r a l i n nature but can be iden t i f i ed a s t he protoplasmic f l u i d i n which the s t r u c t u r a l elements a re embedded. I n addi t ion t o t h e nine components t h a t can be iden t i f i ed i n Figures 1 and 2, muscle c e l l s may also contain membranes of the Golgi complex, lysosomes, and l i p i d inclusions. a l l these s u b c e l l u h r components can be ident i f ied i n e lectron micrographs of i n t a c t muscle c e l l s , but it i s evident t h a t determining the biochemical functions of these subcel lular components depends on i s o h t i o n of each

With the possible exception of lysosomes,

Fig. 1. This electron micrograph of an intermediate type r a t diaphragm muscle f i b e r shows four of the nine o r ten d i f f e ren t components t h a t const i tute muscle c e l l s . P a r t of the nucleus seen i n the lower l e f t - hand corner of the micrograph i l l u s t r a t e s the very large s i ze of th i s subcellular organelle. the nucleus (M ) and scat tered elsewhere i n the c e l l . which a re a sutfcellular organelle unique t o muscle f ibers , course almost ve r t i ca l ly i n t h i s micrograph (Z-disk of one myofibril indicated by Z ) . be distinguished i n the upper left-hand corner of t he micrograph. X13,500. and The University of Wisconsin Press, Madison; 6 1970 by the Regents of the University of Wisconsin, p . 111.

Numerous mitochondria can be seen j u s t above Myofibrils,

The outer c e l l membrane, or sarcolemma, can ju s t

Reproduced frm Gauthier (1970) by permission of the author

253

Fig. 2. The membranous subcel lular components of a muscle c e l l a r e evident i n t h i s e lectron micrograph of f rog (Rana pipiens ) sa r to r ius muscle. Areas indicated by T a re t r i a d s and show various aspects of the T-tubule system t h a t passes t ransversely t o t h e long ax is of t h e muscle c e l l . The presence of large membrane-enclosed sacs cal led l a t e r a l c is ternae on e i t h e r s ide of t he T-tubule i s responsible for t he name, tr iad. Note t h a t t r i a d s i n t h i s muscle occur a t t he l e v e l of every Z-disk. L. designates t h e sarcoplasmic r e t i c u l a r medranes that run p a r a l l e l with the long axis of t h e c e l l . a r e analogous t o the endoplasmic reticulum of nonmuscle c e l l s . Myofibrils a l s o course almost v e r t i c a l l y i n t h i s micrograph. darkly s t a h i n g spher ica l granules a r e glycogen. c o l l a r . of t he author and the University of Wisconsin Press, Madison; 1970 by the Regents of t h e University of Wisconsin, p . 283.

These membranes

Smll,

Reproduced from Peachey (1970) by permission Feenes t r a t ed

Line is 1 w.

254

of these components i n a pur i f ied form. the outer c e l l membrane be broken, and the individual components be separated from t h e r e su l t i ng mixture. Although it is not d i f f i c u l t t o rupture c e l l membranes, it was v i r t u a l l y impossible before development of modern high-speed re f r igera ted centrifuges t o separate the complex mixture of substances produced by such rupture. of high-speed centr i fuges, however, it was c l ea r that simply l e t t i n g c e l l homogenates (i.e., suspensions of subcel lular components a f t e r the outer c e l l membrane had been ruptured) stand under t h e influence of gravi ty caused some separation of t he subcel lular components because the l a rges t components s e t t l e d out af the homogenate rapidly and could be p a r t l y separated from t h e other components by decanting the super- natant solut ion. If the supernatant solut ion from this f i r s t decanting w a s allowed t o set f o r a s l i g h t l y longer period under the influence of gravity, t he next l a rges t component s e t t l e d out and could a l s o be obtained i n a p a r t l y pur i f ied form by again decanting the supernatant. This procedure could be repeated w i t h successively longer s e t t l i n g periods t o obtain p a r t l y pur i f ied preparations of successively smaller p a r t i c l e s (Figure 3). t o obtain p a r t l y pur i f ied preparations of t h e l a rges t subcel lular components, the time required f o r t h e smallest subcel lular p a r t i c l e s t o s e t t l e out of t h e homogenate was prohibi t ively long (indeed, severa l centur ies would be required f o r some of the smallest subcel lular p a r t i c l e s t o s e t t l e out of a homogenate), and it was impossible t o obtain preparations of the smallest subcel lular preparations by using th i s s e t t l i n g procedure.

Such i so l a t ion requires t h a t

Even before development

Although this procedure enabled ea r ly invest igators

Sedimentation of Subcellular Components i n t h e Centrifuge

It soon became evident that separation of subcel lular components produced by s e t t l i n g in a g rav i t a t iona l f i e l d could be accelerated tremendously by increasing t h e force causing s e t t l i n g or sedimentation of the pa r t i c l e s , and that this force could conveniently be increased by spinning c e l l homogenates i n a centr i fuge. Spinning a t low speeds f o r b r i e f periods of time generates low sedimentation forces, and p a r t l y pur i f ied preparations of t h e heaviest subcel lular components can be obtained w i t h only slight contamination by the l i g h t e r subcel lular components. By increasing t h e speed, grea te r sedimentational forces a r e developed, and even the l i g h t e s t subcel lular components can be sedimented i n an acceptable period of time. showed t h a t sedimentation of p a r t i c l e s due t o cent r i fuga l forces obeyed t h e same physical l a w s as sedimentation of p a r t i c l e s due t o g rav i t a t iona l forces , and that differences i n r a t e s of sedimentation of two pa r t i c l e s i n t h e same solvent o r suspending medium depended only on differences i n t he s ize , shape, and densi ty of t he two p a r t i c l e s ( t ab le 1). Because of the d i r e c t r lea t ionship described e a r l i e r between sedimentat ion of subcel lular components i n a cent r i fuga l f i e l d and t h e d i f f e r e n t i a l s e t t l i n g of components i n a g rav i t a t iona l f i e ld , separation of subcel lular components by a s e r i e s of centr i fugat ion cycles each done a t d i f f e ren t speeds is ca l led d i f f e r e n t i a l centrifugation, and the force developed durlng centr i fugat ion i s commonly given i n terms of how many times grea te r it is than t h e force of gravity; e.a., 2000 times gravi ty or, f o r short, 2000 x 8.

Theoretical calculat ions

255

a B C D E TIWE

___F

Fig. 3. Diagrammatic representation of how pirticles of different sizes are separated by differential centrifugation. Initially, a l l particles are distributed uniformly through the centrifuge ( A ) . As centrifugation proceeds (B), all particles sediment at their respective sedimentation rates, resulting first in complete sedimentation of the largest particles present (C), and then in sedimentation of progressively smaller particles (D and E). degree of contamination of larger particles in the pellet by smaller particles at the exact moment when the larger particles have just been completely sedimented (e .a ., see C) is approximately proportional to the ratio of the sedimentation coefficients of the t w o particles. Distribution of particles in tube E is shown in the bar graph at the right. Reproduced from Anderson (1966) by permission of the author and the United States Departnrent of Health, Education and Welfare.

The

TABLE 1

Differences i n r a t e of sedimentation of two p a r t i c l e s

depend on 1. Size 2. Shape 3. Density of t he two p a r t i c l e s

2 2 v = - = dr 2a (Pp-Ps)W r d t

where v = veloc i ty a = p a r t i c l e radius p = density w = angular ve loc i ty r = radial dis tance from center of ro t a t ion i = viscos i ty

f / fo = f r i c t i o n a l coef f ic ien t

As shown i n table 1, veloci ty of sedimentation increases w i t h the Consequently, square of the ro t a t iona l speed of t he centrif'uge ro to r .

much emphasis has been placed on obtaining the grea tes t possible centri- fugal speeds t o permit sedimentation of the smallest pa r t i c l e s , and centrifuges capable of speeds up t o 75,000 r p m a re now commercially ava i lab le . Indeed, it is not d i f f i c u l t t o construct centrifuges capable of speeds i n excess of 100,000 rpm, but it has been irnpossible t o f i n d metals o r metal a l loys of su f f i c i en t s t rength t o bui ld ro to r s capable of withstanding the tremendous forces developed a t these very high speeds. avai lable ro to r i s t rave l ing faster than the muzzle ve loc i ty developed by most high-velocity r i f l e s , and a force s l i g h t l y over 5OO,OOO x e; i s developed. A t t h i s force, one gram of metal i n t h e ro to r "weighs" 'jO0,OOO $rams ( i . e . , a cen t r ipe t a l binding force of a t l e a s t 5O0,OOO grams i s required t o keep the ro to r together) , and a 170-pound man would weigh 85,000,000 pounds o r 42,500 tons--imagine your l egs t ry ing t o support that! a re usual ly made of t i tanium al loys, and cost i n excess of $2000.

A t 75,000 rpn, the outer radius of t h e commercially

Rotors capable of withstanding these very high forces

The equation i n t ab le 1 a l s o shows that sedimentation veloci ty of a p a r t i c l e is p r o p a r t i o n a l t o t h e difference between densi ty of the p a r t i c l e and density of t h e solvent i n which t h e p a r t i c l e is suspended. Solvents used i n d i f f e r e n t i a l centr i fugat ion general ly a r e d i l u t e aqueous solut ions w i t h dens i t i e s only s l i g h t l y g rea t e r than 1.0. Because most subcel lular p a r t i c l e s have dens i t ies between 1.15 and 1.75, the difference i n densi ty between subcel lular p a r t i c l e s and t h e c m o n

257

solvents var ies only f ivefold, whereas t h e difference i n radius of t he same subcel lular p a r t i c l e s ranges from 20 t o 5000 nm or 250-fold. Therefore, a t t he solvent dens i t ies comonly used i n d i f f e r e n t i a l centrifugation, r a t e of sedimentation of W r t i c l e s depends pr inc ipa l ly on p a r t i c l e s i z e and shape, and l e s s on p a r t i c l e densi ty . dependence on p a r t i c l e s i z e and shape i s cha rac t e r i s t i c of the d i f f e r e n t i a l centr i fugat ion procedure t h a t we have been describing. A second cent r i fuga l procedure, ca l led densi ty gradient centr i fugat ion, has a l so been developed and w i l l be described i n more d e t a i l l a t e r in t h i s review. One of t he two d i f f e ren t kinds of densi ty gradient cen t r i - fugation separates p a r t i c l e s so l e ly on the bas i s of t h e i r densi ty . Consequently, a combination of d i f f e r e n t i a l centr i fugat ion, which separates p a r t i c l e s on the bas i s of their s i z e and shape, with the pa r t i cu la r kind of densi ty gradient centr i fugat ion that separates pa r t i c l e s so l e ly on the basis of t h e i r density, provides a high- resolut ion technique f o r pur i f ica t ion of subcel lular p a r t i c l e s . of t h i s technique t o i s o l a t e homogeneous f rac t ions of muscle membranes w i l l be discussed l a t e r i n t h i s review.

This

Use

Factors Affecting Sedimentat ion Coefficients of Subcellular Components

The a v a i l a b i l i t y of re f r igera ted ul t racentr i fuges capable of speeds up t o 75,000 rpm has made it possible t o sediment even r e l a t i v e l y small protein molecules from c e l l homogenates. Consequently, sedlmentation ve loc i t i e s , expressed as ve loc i ty per un i t of centrif 'ugal accelerat ion so t h a t they a r e independent of t h e speed used t o sediment t he pa r t i c l e , have been determined f o r many subcel lular m q o n e n t s . sedimentat. n coef f ic ien ts , expressed i n Svedberg u n i t s (one Svedberg u n i t = sec) , are l i s t e d i n t a b l e 2. It is evident from the da ta i n t a b l e 2 t h a t s i z e is the predominant fac tor determining sedimentation coef f ic ien ts of subcel lular cmponents i n d i f f e r e n t i a l centr i fugat ion. Almost without exception, l a rge r components have grea te r sedimentation coef f ic ien ts than smaller components, and there is no obvious ordering of sedimentation coef f ic ien ts according t o densi ty of t h e components L c f . t a b l e 9 ) . than microsomes o r plasma membranes (cf. t a b l e g ) , but RNA, DNA, and ribosanes a l l have smaller sedimentation coef f ic ien ts than microsomes o r plasma membranes because RNA, DNA, and ribosomes a re smaller than microsomes o r plasma menibranes. is given f o r each of the components l i s t e d i n t a b l e 2. mitochondria, lysosomes, RNA, DNA, and proteins , t h i s range of sedi- mentation coef f ic ien ts probably indicates t h a t these components e x i s t i n a range of s i zes i n l i v i n g c e l l s . considerably i n s i z e and when only muscle c e l l s a r e considered, the range of sedimentation coef f ic ien ts l i s t e d f o r polysomes i n t a b l e 2 is too small because t h e polysomes that code f o r myosin synthesis a r e among the l a rges t polysomes known and have a sedimentation coef f ic ien t i n the range of lOOO-5OOO S. On t h e other hand, t he wide va r i e ty of d i f f e ren t s i zes of myofibrils, plasma membranes, and microsomes that e x i s t s i n a muscle c e l l homogenate i s produced during homogenization of t he c e l l ,

Some of these

For example, RNA, DNA, and ribosomes are a l l much denser

A range of sedimentation coef f ic ien ts For nuclei ,

For example, polysomes range

TABU 2. APPROXIMATE SEDINEXI"TT1ON COEFFICIENTS OF MAJOR COMPONENTS OF MAMMALIAN CELLSa

Cel l Component Sedimentation Coefficient ( S )

Soluble proteins

RNA

DNA

Ribosomal subunits

Ribosomes

Polysomes

Glycogen

Microsomes

Plasma menibranes

Lys os one s

Mitochondria

Myof i b r i Is

Nuclei

1 - 25

4 - 50

up t o 100

30 - 60

70 - 80

100 - 400

100 - 10,000 100 - 15,000 loo - 100,000

10,000 - 20,000

20,000 - 70,000

100,000 - 4,000,000 ( ? )

4,000,000 - 10,000,000

a Sedimentation coeff ic ients a r e expressed i n Svedberg uni t s where one Svedberg = lO-l3 sec.

and s izes of these components i n muscle c e l l homogenates a re not indi- cat ive of t h e i r s i z e i n t h e l iv ing c e l l . electron micrographs of i n t ac t muscle c e l l s tha t myofibrils and the sarcoplasmic r e t i cu la r and T-tubule membranes that const i tute the microsomal f rac t ion i n homogenates a re very extensive i n s i t u and ac tua l ly extend unbroken throughout the i n t a c t c e l l . Homogenization, and consequent rupture, of the outer c e l l membrane produces fragments of the plasma membrane and a l so ruptures the myofibrils, T-tubules, and sarcoplasmic r e t i c u l a r membranes inside the c e l l . The round, membrane- enclosed vesicles that const i tute the microsomal f rac t ion i n muscle homogenates (see f igures 11 and 12) a re formed by sealing of the broken ends of ruptured sarcoplasmic r e t i cu la r o r T-system membranes due t o surface tension forces generated by exposure of hydrophobic membrane

Indeed, it i s obvious from

259

l ipoproteins t o an hydrophilic homogenizing solvent . It i s c l ea r , therefore , that s i z e of t he pa r t i c l e s i n myofibril, plasma membrane, and microsomal f r ac t ions is re la ted d i r ec t ly t o the kind and duration of homogenization used t o rupture the outer c e l l membrane; a mild or shor t homogenization w i l l r e s u l t i n large p a r t i c l e s with large sedi- mentation c z f f i c i e n t s i n these f rac t ions , but severe or prolonged homogenization w i l l cause very small p a r t i c l e s with small sedimentation coef f ic ien ts i n t he same f r ac t ions . I f homogenization i s severe or prolonged, other subcel lular compctnents such a s nuclei , mitochondria, o r lysosomes may a l s o be damaged, and these damaged components w i l l not sediment normally. Consequently, the homogenization procedure used t o rupture c e l l s has important e f f ec t s on sedimentation propert ies of subcel lular components during d i f f e r e n t i a l centrifugation, and homo- genization conditions should always be specif ied when describing f rac t iona t ion of subcel lular components by d i f f e r e n t i a l centr i fugat ion.

I n addi t ion t o homogenization procedure, the type of centrifuge ro tor or head can a l s o have pronounced e f f ec t s on separation achieved by d i f f e r e n t i a l centr i fugat ion. may be used f o r d i f f e r e n t i a l centr i fugat ion: (1) an angle-head or conical ro tor i n which tubes a re incl ined a t a f ixed angle t o the horizontal plane ( f igure 4); and (2) a swinging bucket or swing-out r o t o r i n which tubes a r e suspended from a ro ta tab le pin and a r e f r e e t o swing out and assume a per fec t ly horizontal posi t ion during ro t a t ion ( f igure 4 ) . Thus f a r , it has not been possible t o construct swinging bucket ro to r s capable of swinging large volumes a t very high speeds, and most d i f f e r e n t i a l centr i fugat ion has been done with angle- head ro to r s . Fortunately, it has been shown (Anderson, 1968; Pickels, 1943; Schumaker and Rees, 1969) that t h e theory f o r sedimentation of p a r t i c l e s i n a cent r i fuga l f i e l d holds f o r sedimentation i n angle-head ro to r s as wel l as it does f o r sedimentation i n swinging bucket ro to r s . Regardless of t he type of ro tor , sedimentation always occurs r ad ica l ly i n a d i rec t ion perpendicular t o the axis of ro t a t ion (second and four th pa r t s of f igure 4 ) . w a l l i n angle-head ro tors , they p e l l e t there , and the p e l l e t gradually s l i d e s toward t h e bottom of t he tube depending on t h e in te rp lay of f r i c t i o n a l and g rav i t a t iona l forces (Anderson, 1968; Schumaker and Rees, 1969). s l i d i n g of pe l le ted material along the outer inclined tube w a l l i n angle-head ro to r s would produce convective disturbances serious enough t o a f f e c t t he rates a t which p a r t i c l e s sediment r ad ia l ly , more recent results (Charlwood, 1963) have shown t h a t remarkably l i t t l e convection occurs during sedimentation in angle-head ro tors , and that angle-head ro tors may even be used very e f f ec t ive ly f o r densi ty gradient centr i fugat ion (Brentani e t al . , 1967; Fisher e t -- al . , 1964; Martin and Ames, 1961) where convective disturbances have ser ious e f f ec t s . Because sedimentation i n angle-head ro tors does not seem t o be accompanied by large convective movements, t h e p r inc ipa l difference between sedimentation i n angle-head ro to r s and swinging bucket ro to r s is the much shor te r sedimentation dis tances required t o p e l l e t materials a g a i n s t t he aube w a l l in angle- head ro to r s (arrows i n second and four th parts of f igure 4 ) .

Two general kinds of centrifuge ro tors

When sedimenting pa r t i c l e s s t r i k e the outer inclined

Although e a r l i e r invest igators (Pickels, 1943) suggested t h a t

Consequently,

260

ANGLE ROTOR SWINGING BUCKET ROTOR

n

Fig. 4. Schematic representation of sedimentation in angle-head and swinging bucket r o t o r s . ro to r i s shown i n t he tube held a t an angle of 23.50 t o t h e v e r t i c a l (or 6 6 . 5 O t o t he hor izonta l ) the swinging bucket ro to r as it would ap ar when spinning; tubes

both kinds of ro tors , sedimentation proceeds r a d i a l l y as indicated by the arrows.

A cut-away s ide view of the angle-head

A t far r igh t , a top view is shown of

a l l pro jec t d i r e c t l y outward r a d i a l l y (0 ge t o the hor izonta l ) . I n

261

t h e time required t o sediment a given subcel lular component out of a c e l l homogenate w i l l l i k e l y be l e s s when centr i fugat ion i s done i n an angle-head rotor t han when a swinging bucket ro tor i s used. Because of t h i s , the kind of r o t o r used can a l so have important e f f ec t s on the conditions necessary t o obtain optimal separation by d i f f e r e n t i a l centr i fugat ion.

Because ve loc i ty of sedimentation, or what i s equivalent, cen t r i fuga l force developed by the centrifuge ro tor , depends on r a d i a l distance from the center of rokation (see t a b l e l), it therefore follows t h a t cen t r i fuga l force var ies depending on posi t ion of the p a r t i c l e i n t h e centrif'uge tube. For example, a p a r t i c l e near t he bottom of the tube i n a swinging bucket ro to r i s being subjected t o a l a rge r cent r i fuga l force than the same p a r t i c l e would be i f it were near t h e top of the same tube because i t s r a d i a l dis tance f r D m t h e center of ro t a t ion is g rea t e r . These differences can become very large a t high centrifuge speeds (e&., between 180,000 x B and 420,000 x g f o r a commercial ro to r a t 65,000 rpm), and it is therefore important t h a t they be considered when attempting t o do carefu l d i f f e r e n t i a l cen t r i fuga l separation of subcel lular components. Most centrifuge manufacturers publicize only t h e maximum forces obtainable with t h e i r instruments ( i .e . , t h e force a t t h e bottam of t h e tube) t o place themselves i n t he best competitive pos i t ion . When l i s t i n g Centrifugal conditions i n a s c i e n t i f i c publication, however, t h e average cent r i fuga l force (i .e ., t he cent r i fuga l force developed a t a posi t ion halfway between t h e maximum and minimwn radial d is tances) should be given because this i s the average force t o which p a r t i c l e s i n t h a t centr i fuge tube would be subjected. any departure from the procedure of lrPsting the average cent r i fuga l force should be e x p l i c i t l y indicated. f o r l i s t i n g cent r i fuga l forces a t d i f f e ren t posi t ions i n the centrifuge tube i s &, $nin or @;ave; hence 220,000 x hve.

A t t h e very l e a s t ,

The notation customarily used

Separation of Subcellular Components of Muscle Cel ls by Di f f e ren t i a l C e n t r i f w t i o n .

With t h i s b r i e f introduction t o some of t h e important fac tors a f f ec t ing separation of subcel lular components by d i f f e r e n t i a l cen t r i - fugation, t he Use of d i f f e r e n t i a l centr i fbgat ion t o separate subcel lular components from muscle c e l l s w i l l be discussed. severa l unique problems t o the appl icat ion of d i f f e r e n t i a l centr i fugat ion. F i r s t , muscle c e l l s a r e surrounded by a tough basement membrane, 40-70 nm i n thickness, and t h i s basement membrane is i n t u rn connected t o t h e endomysia1 connective t i s s u e layer . makes it very d i f f i c u l t t o rupture the outer c e l l membrane, or sarcolemma, of muscle c e l l s . of muscle c e l l s , pa r t i cu la r ly i n mature muscle, it is necessary t o mince t h e muscle very f i n e l y i n an instrument like a s i l e n t chopper o r t o grind t h e muscle i n a meat gr inder . t he sarcolemma in to small pieces that sediment with the microsomal f rac t ion , and may a l so damage the other la rge subcel lular components such as nuclei

Muscle t i s s u e presents

The presence of these two layers

Consequently, t o obtain reasonably complete d is in tegra t ion

Such mincing or grinding generally fragments

262

or mitochondria. If large, r e l a t i v e l y i n t a c t sarcolemmal sheaths a re desired, t he muscle t i s s u e m u s t be cut i n to small pieces with a sc i ssors , and these pieces homogenized i n a Waring Blender or similar device without subjecting the c e l l s t o severe mincing o r grinding. D r . Cedric Matsushima w i l l probably discuss the procedure necessary t o obtain i n t a c t sarcolemmal sheaths fran muscle t i s sue i n h i s paper l a t e r today. e f f i c i e n t a t rupturing c e l l s from mature muscle c e l l s , but it i s possible t o obtain reasonably complete rupture of embryonic muscle c e l l s simply by using a Potter-Elvejhem or similar homogenizer ( t ab le 3 ) . ruptured without grinding or f i n e mincing. After t h e c e l l s have been ruptured by grinding o r f i n e mincing, a muscle c e l l homogenate is prepared by suspending the ruptured c e l l s i n a su i tab le medium with a Waring Blender o r other similar device ( t ab le 3 ) . suspension of t h e ruptured c e l l s , and a l l subsequent s teps i n separation of subcel lular components by d i f f e r e n t i a l centr i fugat ion should be done a t cold temperatures (1-3' C ) with pre-cooled apparatus and solut ions t o prevent undesired degradation of subcel lular components by endogeneous enzymes and t o s top other metabolic and c e l l u l a r react ions as completely as possible .

Mincing with a sc i s so r s followed by homogenization i s not very

Occasionally, muscle c e l l s from very young animals can a l s o be

Ce l l rupture,

Some recommendations on ingredients t h a t should be included i n t he homogenizing medium used t o suspend ruptured muscle c e l l s and t h e functions of these ingredients a r e given i n t a b l e 4 . which i s t h e most abundant soluble substance i n l i v ing mammalian muscle c e l l s , o r sucrose i s used t o maintain proper osmotic conditions surrounding the l ibera ted subcel lular components. Inclusion of K C 1 o r sucrose is very important, f o r without these substances, mitochondria, nuclei , lysosomes, and microsomal ves ic les w i l l swell and bu r s t . Myofibrils w i l l a l so swel l and lose a l l s t ruc ture if they a r e suspended i n a solut ion of zero osmolarity. I n some instances, t he homogenizing medium is de l ibera te ly made hypertonic t o cause membranous subcel lular components t o shrink s l i g h t l y i n an t ic ipa t ion that the shrunken components w i l l be more r e s i s t a n t t o breakage during homogenization t h a n t h e i r normal o r s l i g h t l y swollen counterparts. included i n the homogenizing medium t o maintain pH of the homogenate near -- i n vivo leve ls ( t a b l e 4 ) . buffer ing capacity a t pH 6.8-7.0, it is general ly t o be preferred over T r i s (abbreviation f o r tris-(hydroxymethy1)-aminomethane) . Phosphate buffer , however, must l a t e r be removed i f the subcel lular component i s t o be assayed f o r any enzymic a c t i v i t y involving re lease of inorganic phosphate (such as assaying myofibrils f o r ATPase a c t i v i t y ) . of a buffer is pa r t i cu la r ly important in studies comparing postmortem muscle, which has a low pH, with muscle obtained immediately after death when it has a pH near 7.0. medium i n such s tudies w i l l r e s u l t i n da ta t h a t a r e hopelessly confounded by the difference i n pH under which the subcel lular f ract ionat ions are done. A Ca2+-chelator and Mg2+ are included t o l i m i t the amount of myofibr i l lar contraction caused by g r i n d i and homogenization. Even i n t h e presence of a Ca2+-chelator and Mg', it is very d i f f i c u l t t o

Generally, KC1,

A small amount of buffer i s

Because phosphate has a much la rger

Inclusion

Omission of buffer from the homogenizing

T A B U 3 . PREPARATION OF MUSCLE TISSUE FOR D I F F E R E N T I A L CENTRIFUGAL SEPARATION OF SUBCELLULAR COMPONENTS

Pr o c e du r e Rat ionale

1. Rupture c e l l s by passing - mature muscle c e l l s cannot be throu& a meat grinder o r other cu t t e r similar homogenizers and must

ruptured by Pot te r -Elve jhem or

be minced or grsund. Meat grinders fragment the sarco- lemma i n t o small pieces.

2 . Suspend ru>tured c e l l s i n - Potter-Elve jhem or similar isotonic or hypertonic homogenizer cari be used f o r median by use of Waring young muscle. Type of medium Blender o r similar device used and sever i ty OP homogeni-

zation can have important e f f ec t s on the way subcel lular components sediment during d i f f e r e n t i a l centr i fugat ion.

TABLE 4. INGREDIENTS I N MEDIUM USED TD SUSPEWD BROKEN MUSCLE CELLS P R I O R TO D I F F E R E N T I A L CENTRIFUGATIONa

Ingredient Purpose

1. 0.25 t o 0.8 M sucrose o r 50 t o 150 mM K C 1

- maintain osmDtic re la t ionships t o prevent disrupt ion of organelles.

2 . 10 t o 50 mM T r i s o r - maintain pH a t near i n vivo leve ls t o K-phosphate buffer , insure reproducible centr i fugal PH 6.8-7.4 separations.

3 . 1 t o 5 mM EGTA o r EDTA - chelate Ca2+ and e l e t e ous heavy metals such as ??e$+, Cu”, e t c . Ca2+ chelation helps t o l i m i t extent of contraction during homogenizatiDn.

4 . 1 t o 5 M g C 5 - help prevent actin-myosin in te rac t ion and contraction during homogenization. Use only with EGTA.

a These ingredients are recommendations for general purpose d i f f e r e n t i a l centr i fugal of muscle c e l l s and d i f f e ren t ingredients may be necessary f o r spec i f i c purposes.

264

prepare myofibrils completely i n the relaxed s t a t e from muscle c e l l s obtained immediately a f t e r death of t he animal. and Berthet, 1954) have indicated that, i n l i v e r c e l l homogenates, chelation of Ca2+ enhances mitochondrial s t a b i l i t y , but t h a t a t r a c e of Ca2+ i s necessary f o r p r e p r a t i o n of i n t a c t nuclei by using d i f f e r e n t i a l centr i fugat ion. To our knowledge, these asser t ions have not been t e s t ed i n muscle c e l l homogenates. Obviously, because it chelates both Ca2+ and Mg2+, EDTA (abbreviation f o r ethylenediamine t e t r a a c e t i c ac id) should not be included i n a homogenizing medium i f Mg2+ i s a l s o included. A s noted i n t ab l e 4, the presence of EDTA o r EGTA (abbrevlaticm f o r 1,2-bis - -( 2-dicarboxymethylaminoethyoxy ) -ethane ) i n t h e homogenizing medium has the addi t iona l bene f i c i a l e f f e c t of chelating t r a c e anr>unts of heavy metal contaminmts t h a t may be i n t h e hmogenizing medium or be released from the grinder or homogenizer, and t h a t generally have deleter ious e f f ec t s on subcel lular components.

Some reports (de Duve

A summary of the cent r i fuga l conditions that may be used t o f rac t iona te muscle c e l l homogenates i n t o myofibr i l lar plus nuclear, mitochondrial, lysosomal, microsomal and sarcoplasmic f r a c t i m s is given i n t a b l e 5. This t a b l e i l l u s t r a t e s the second unique problem encountered when attempting t o f rac t iona te muscle c e l l h m g e n a t e s by d i f f e r e n t i a l centrifugation; muscle c e l l s contain myofibrils, a sub- c e l l u l a r component not possessed by o ther c e l l s , and myofibrils sediment with nuclei . nuc le i not heavi ly contaminated with myofibrils by using d i f f e r e n t i a l centr i fugat ion alone. On the other hand, myofibrils r e l a t i v e l y f r e e from nuclei can be obtained simply by subjecting the myofibr i l lar f r ac t ion t o repeated homogenization in t h e Maring Blender. genization ruptures the nuclear membrane, and the fragmented membrane and nuclear contents are not sedimented a t low cent r i fuga l forces . The conditions of cent r i fuga l speed and time l i s t e d i n t ab le 5 a r e f o r homogenizing times of 10-20 seconds and a homogenizing medium similar t o t h a t given i n t ab le 4. longer homogenizing times a r e used, cen t r i fuga l speeds o r t ims or both w i l l need t o be increased t o obtain adequate separation. t he converse would apply i f shor te r homogenizing times a re used. Also, i f only one of t h e f i v e f rac t ions l i s t e d i n t ab le 5 is desired, the cent r i fuga l boundary conditions given as necessary t o sediment that f rac t ion might be widened t o increase the y ie ld of that pa r t i cu la r f r ac t ion a t the expense of pu r i ty of t h e f r ac t ion and y ie lds of the bordering f r ac t ions . Although t h e 100,000 x g supernatant i s l i s t e d as t h e final f r ac t ion i n t a b l e 5, it i s possible, with modern preparative u l t racent r i fuges , t o sediment proteins and high molecular weight substances out of this supernatant. For example, any pro te in o r other p a r t i c l e having a sedimentation coef f ic ien t of 5s or grea te r can be sedimented from the 100,OOO x g supernatant by centrifuging f o r 5-8 hours a t 75,000 rpm (500,000 x 8)

Therefore, it i s very d i f f i c u l t t o obtain muscle

Such homo-

If a more viscous homogenizing medium or i f

O f course,

TABU 5 . SEPARATION OF SUBCELIULAR COMPONENTS OF THE MUSCLE CELL BY DIFFJBEDTUL CENTRIFUGATION^

Conditions Components Sedimented

0 t o 2000 x g f o r 10-15 min

2000 t o 10,000 x g f o r 10-20 min

10,000 t o 25,000 x g fo r 20-30 min

25,000 to 100,000 x g f o r 120-l-80 m i n

100,000 x g supernatant

- Sediments nuclei, myofibrils, unbroken ce l l s , connective t i s sue , and sarcolemmal sheaths.

- Sediments mitochondria

- Sediments lysosomes and “heavy” microsomes

- Sediments microsomes, polysomes , ribosomes and fragments of sarcolemma

- Sarcoplasmic proteins, t-RNA, and other soluble substances.

a These conditions a re useful for complete f ract ionat ion of a muscle Different conditions may be more appropriate f o r c e l l homogenate.

separation of a spec i f ic component.

Because myofibrils a r e the cont rac t i le elements of muscle and because preparation of myofibrils i s often the f i rs t s t ep i n purif icat ion of one o r more of t’ne myofibril lar proteins, myofibrils a re probably the subcellular component most frequently prepared from muscle c e l l homogenates by d i f f e ren t i a l centrifugation. Although a large number of d i f f e ren t procedures have been described i n the l i t e r a t u r e f o r preparation of myofibrils, several important improvements i n these procedures have been made recently. our laboratory has developed f o r preparation of highly purif ied myofibrils. s t r a i n e r i n Steps I11 and IV and solubi l izat ion of membranes adhering t o the myofibril lar surface i n Steps V and V I a re par t icu lar ly important features of this procedure. We have found t h a t a common household nylon s t r a ine r is the most e f f i c i e n t device avai lable f o r remving connective t i s sue from myofibrils. Treatment with 1% Triton X - 1 0 0 ( s teps V and V I ) has no detectable e f f ec t on the biological o r s t ruc tu ra l properties of the myofibril lar proteins but almost completely removes the membranes that normally adhere t o the surface of myofibrils. Becauee these membranes contain a Mg2+-mdified ATPase ac t iv i ty , it is important t h a t they be absent fram myofibril lar preparations t h a t w i l l be assayed fo r ATPase ac t iv i ty .

Table 6 shows a procedure t h a t

Remow1 of connective t i s sue by passing through a nylon

266

(contains CAF and other sarcoplasmic proteins )

TABLE 6 . PREPARATION OF PURIFIED MYOFIBRILS BY USING DIFFlERENTIAL CENTRINGAT ION

1)Suspend i n 6 volumes (v/w ) of standard s a l t solut ion by homogenizing f D r 10 sec i n Waring blender.

2)Centrifuge a t 1000 x g for 10 min.

(d iscard)

Supernat a n t (d i scard)

1 j

1)Suspend i n 8 volumes (v/w) of standard sal t solut ion by homogenizing f o r 10 sec i n Waring blender.

2 )Pass suspension through household nylon net s t r a i n e r . 3 )Centrifuge a t 1000 x g f o r 10 min .

1)Suspend i n 8 volumes (v/w) of standard salt solut ion by homogenizing f o r 10 sec i n W a r i n g blender.

2)Pass suspension through household nylon net s t r a ine r . 3 )Centrifuge a t 1000 x g f o r 10 min . 1

(discard) 1)Suspend i n 6 volumes (v/w) of standard sal t solution plus 1% (v/w) Tri ton X - 1 0 0 by homogenizing f o r 10 sec i n Waring Blender.

2 ) ~ e n t r i f u g e a t 1500 x f o r 10 min.

Supernat an t (d i scard) 1)Suspend i n 6 volumes (v/w) of standard sal t solut ion

plus 1% (v/w) Tri ton X - 1 0 0 by homogenizing f o r 10 sec i n Waring blender.

2)centrifuge a t 1500 x g f o r 10 min.

Supernatant (discard) 1)Suspend i n 8 volumes (v/w) of standard sal t solut ion

2)Centri-e at 1500 x g f o r 10 min. by s t i r r i n g vigorously with polyethylene s t i r r i n g rod.

Supernatant (d i scard) 1)Repeat Step VI1 four times, but suspending i n 8

volumes (v/w) of 100 mM K C 1 instead of standard sal t solutions; centrifuge a t 1500 x g a f t e r each

volumes (v/w) of 100 mN K C 1 by f o r 3 ser i n Waring blender.

(d i scard) 1)Suspend i n 8 homogenizing

Supernatant (d i scard) 1)Suspend i n 2 volumes (v/w) of 100 mM K C 1 by

homogenizing f o r 3 sec i n Waring blender. 2)Do protein analysis of suspension.

268

FRACTIONATION OF SUBCELLULAR ORGANELLES BY DENSITY GRADIENT CENTRINGATION

Although d i f f e r e n t i a l centrifugation is an invaluable method f o r separation of crude whole c e l l homogenates in to fract ions containing pa r t i c l e s of the same s ize , it i s almost impossible by using d i f f e ren t i a l centrifugation alone t o obtain these pa r t i c l e s i n highly purif ied form. Closer examination of f igure 3 shows why t h i s i s so. I n d i f f e r e n t i a l centrifugation, a l l subcellular components a r e dis t r ibuted uniformly throughout t he centrifuge tube a t t he beginning of t he fract ionat ion procedure ( l e f t tube, f igure 3 ) . the la rges t pa r t i c l e s i n i t i a l l y a t the top of the tube a l l t he way t o the b o t t m of the tube (tube C y f igure 3) , the small par t i c l e s t ha t i n i t i a l l y were near the bottom of the tube have also been pelleted because, even though the sedimentation coeff ic ients of these small par t - icles a re far l e s s than that of the large pa r t i c l e s , they have a much shorter distance t o t r a v e l before they a re pel le ted. from the equation in Table 1 that if the sedimentation coeff ic ient of the heavier pa r t i c l e s is three times Larger than that of the smaller pa r t i c l e s , required t o sediment lo@ of the heavier pa r t i c l e s . mentation coeff ic ient of the heavier pa r t i c l e s i s t e n times greater t h a n t h a t of the smaller pa r t i c l e s , 1346 of the smaller pa r t i c l e s w i l l be sedimented i n the time required t o p e l l e t lo@ of the heavier pa r t i c l e s . Although the amount of contaminating, slowly sedimenting pa r t i c l e s present in a f rac t ion can be reduced by resuspending the pe l l e t and resedimenting the suspension, such a procedure i s very time consuming and subjects the subcel lular component t o undesirable addi t ional homo- genization and handling. Moreover, complete pur i ty of t he d e s b e d subcellular component i s approached asymtotically when using repeated cycles of d i f f e r e n t i a l centrifugation, and many cycles of centrifugation and suspension a re required t o obtain 9 6 pur i ty o r greater f o r f ract ions t h a t contain contaminants with sedimentation coeff ic ients very s imilar t o t h a t of the main component. This s i t ua t ion i s exacerbated when the same subcellular components ex i s t i n d i f f e ren t s izes and have a range of sedimentation coeff ic ients (see table 2 and e a r l i e r discussion i n this review). t o obtain purif ied f rac t ions by d i f f e r e n t i a l centrifugation has been investigated theore t ica l ly (Schumaker and Rees, l969), d i f f e r e n t i a l centrifugation c l ea r ly is not very usefu l f o r purifying subcellular pa r t i c l e s after t h e i r i n i t i a l f ract ionat ion from a crude c e l l homogenate.

D u r i n g the time required t o sediment

It can be calculated

of the smaller pa r t i c l e s w i l l be sedimented i n the time Even i f the sedi-

Even though the optimal combination of conditions necessary

Because preparations of subcellular pa r t i c l e s must be as pure as possible f o r t h e i r biochemical functions in a c e l l t o be assayed accurately, intensive e f f o r t s were made t o develop sa t i s fac tory procedures f o r addi t ional pur i f ica t ion of the p a r t l y purif ied f rac t ions obtained from crude c e l l homogenates by d i f f e r e n t i a l centrifugation. I n 1951, Brakke, (Brakke, 1951, 1953) a t the University of Nebraska introduced the idea of centrifugation i n a density gradient, and t h i s technique has now been expanded and developed u n t i l densi ty gradient Centrifugation i s t h e couunonly accepted procedure f o r pur i f ica t ion of subcellular organelle

f rac t ions obtained by d i f f e r e n t i a l centr i fugat ion of crude c e l l homogenates. It should be c l e a r l y understood that i n s p i t e of t h e development of zonal ro tors and zonal ul t racentr i fuges designed t o accomodate the l a rges t possible samples (Anderson, 1%6), high- resolution, density gradient centr i fugat ion i s inherent ly l imited t o r e l a t i v e l y s m a ~ samples ( f o r example, even zonal ro tors cannot accommodate samples l a rge r than 100 m l without loss of reso lu t ion) . Because d i f f e r e n t i a l centr i fugat ion, pa r t i cu la r ly a t low cent r i fuga l speeds, can e a s i l y be applied t o samples of two l i t e r s or more, densi ty gradient centr i fugat ion i s most appropriately used t o purify the p a r t l y pur i f ied f rac t ions obtained by d i f f e r e n t i a l centr i fugat ion of crude c e l l homogenates . as much as two l i t e r s of muscle c e l l homogenate by d i f f e r e n t i a l centr i fugat ion ( t ab le 5 ) can e a s i l y be resuspended i n 10-20 ml, and t h i s l a t t e r volume can be r ead i ly accmoda ted by densi ty gradient centr i fugat ion.

Hence, the ''lysosomal f rac t ion" obtained from

Categories of D e n s i t y Gradient Centrifugation

A s a r e s u l t of the intense development of density gradient cen t r i - fugation since i t s inception i n 1951 (de Duve e t -- a l . , 1959), a t l e a s t two general categories of density gradient centr i fugat ion can now be distinguished. i n the centrifuge tube, the purpose of the gradient and t h e physical bas i s underlying separation of the subcel lular components i n the two categories a re completely d i f f e ren t . The first category i s the r a t e - zonal method, f requent ly referred t o simply a s t he zonal method. The e s s e n t i a l fea tures of t h i s method a re i l l u s t r a t e d i n f igure 5 . The densi ty gradient i n zonal densi ty gradient centr i fugat ion i s preformed and is represented by the gradual increase i n shading between the t o p and bottom of the tubes i n f igure 5 . Density gradients i n zonal density gradient centr i fugat ion a re commonly made of sucrose because sucrose i s inexpensive and readi ly avai lable , but other gradient mater ia ls may a l s o be used (see t ab le 8 and subsequent discussion) . The pa r t ly pur i f ied sample of subcel lular components obtained a f t e r sedimentation by d i f f e r e n t i a l centr i fugat ion and resuspension i n a small amount of the appropriate solvent i s careful ly layered i n a t h i n zone on top of t he densi ty gradient (hence, the name zonal centr i fugat ion, f igure 5 ) . The densi ty gradient i n zonal centr i fugat ion is used pr inc ipa l ly t o support and s t a b i l i z e the zone of layered mater ia l . t o suspend samples destined f o r zonal centr i fugat ion should not be denser than t h e t o p of the densi ty gradient o r the sample w i l l s ink in the gradient before Centrifugation i s s t a r t e d with r e su l t i ng mixing and spreading of t he zone. During centr i fugat ion, the d i f f e ren t subcel lular components present i n t he s t a r t i n g zone sediment through the densi ty gradient at r a t e s determined primarily by t h e i r s i z e and shape; these a r e the same fac to r s t h a t a f f e c t r a t e of sedimentation i n d i f f e r e n t i a l centr i fugat ion. I n zonal centr i fugat ion, however, a l l subcel lular components a re i n a t h i n zone a t t h e top of the tube when centr i fugat ion begins ra ther than being d is t r ibu ted uniformly throughout the tube as

Although both these categories employ a densi ty gradient

Therefore, the solvent used

F3.g. 5. A schematic diagram showing separation of p a r t i c l e s by r a t e - zonal densi ty gradient centr i fugat ion. samples a re i n i t i a l l y layered i n a t h i n zone over t he density gradient (Tube A). have sedimented d i f f e ren t dis tances Fnto the gmdient ; the distance the p a r t i c l e s a r e sedimented is a m c t i o n pr inc ipa l ly of the s i z e and shape of t h e pa r t i c l e s , and density of the p a r t i c l e s i s of l e s s e r importance (note that l a rge r p a r t i c l e s have sedimented f a r t h e r t h a n medium-sized p a r t i c l e s which have sedimented f a r t h e r t h a n small p a r t i c l e s i n the i l l u s t r a t i o n i n Table B ) . The densi ty gradient i n rate-zonal centr i fugat ion is used t o s t a b i l i z e t h e i n i t i a l and the sedimenting zones r a the r than cause separation on the basis of densi ty . author and Beckman Instruments, Inc .; 6 1965 by Beckman Instruments, Inc .

I n rate-zonal centrifugation,

After centr i fugat ion (Tube B ) , the d i f f e ren t p a r t i c l e s

From Anderson (1965) and r e p oduced by persmission of the

271

TABLE 7. CATmRIXS OF DENSITY GRADIENT CJ3NTRIFUGATION

Type of densi ty gradient centr i fugat ion Character i s t i c s

1. Rate--zonal o r zonal c e n t r i f ugat ion

- gradient i s preformed and components t o be separated a r e layered on t o p of t he gradient . Purpose of the gradient i s t o prevent convection during centr i fugat ion. Different components sediment as bands o r zones moving a t d i f f e ren t r a t e s through the densi ty gradient. Centrifugation may be a t low o r high forces but generally i s not - done f o r long (>4-5 h r ) periods of time. Separation depends on s i ze , shape, and dens i ty of components.

2 . Isopycnic--zonal o r - gradient may be preformed and the sample isopycnic cent r i fu- layered on it or sample and gradient ga t ion m t e r i a l m a y be dispersed uniformly through

the tube. high speeds (up t o 75,000 rpm) and f o r long periods of time (24 hr or longer). Components a r e banded i n the gradient a t a point equal t o t h e i r densi ty and separation depends only on densi ty .

Centrifugation i s done a t very

they a re a t t h e start of d i f f e r e n t i a l centr i fugat ion. Consequently, a l l p a r t i c l e s must t r a v e l t h e same dis tance t o be sedimented t o the bottom of the tube. completely f r e e from more slowly sedimenting p a r t i c l e s ( f igu re 5 ) . procedure may be contrasted w i t h d i f f e r e n t i a l centr i fugat ion where some slowly sedimenting p a r t i c l e s i n i t i a l l y near t h e bottom of t h e tube m u s t t r a v e l only a small distance t o be pe l le ted and a re therefore unavoidably sedimented w i t h l a rge r p a r t i c l e s (cf. Figures 3 and 5 ) . separations a r e normally run u n t i l the heaviest components have sedimented near ly t o t h e bottom of the tube. t he d i f f e ren t sedimenting components is obtained. Most zonal centr i fugat ion a r e done i n swinging bucket ro to r s because it w a s thought that sedimentation i n angle-head ro tors was accanpanied by ser ious convective distances that would d is rupt t h e gradient . the most recent information s h o w s t h a t sedimentation i n angle-head ro to r s occurs r e l a t i v e l y f r e e from convective movements, and severa l studies have shown that high resolut ion zonal centr i fugat ion separations can be done i n angle-head ro to r s (Fisher e t al., 1964; Martin and Ames, 1961). S w i n g i n g bucket ro to r s have contfnued t o be popular f o r zonal centr i fugat ion because they general ly provide a longer path length f o r sedimentation than

This allows f a s t e r sedimenting p a r t i c l e s t o be obtained T h i s

Zonal centrifugation

A t this point, maximum reso lu t ion of

As indicated e a r l i e r i n t h i s review, however,

angle-head ro tors ( f igure 4), and grea te r path lengths produce grea te r resolut ion between s imi la r ly sedimenting components. that although zonal centr i fugat ion is considered a form of density gradient centrifugation, densi ty of the cmponents i s not a major f ac to r a f fec t ing t h e i r separation.

It might be noted

The preformed gradients i l l u s t r a t e d i n f igure 5 can be prepared i n a va r i e ty of ways. can be used t o prepare l i n e a r densi ty gradients . I n t h i s device, two vesse ls of i den t i ca l inside diameter are placed a t i den t i ca l heights on two magnetic s t i r r e r s and connected a t t h e i r base with a piece of tubing. The tubing connecting t h e two vessels and the ou t l e t tubing from the f ron t vessel ( i . e . , the vesse l t o the r i g h t i n f igure 6 ) a r e both clamped shut, and the mater ia l t h a t w i l l cons t i tu te the densest pa r t of the gradient (e.g., 5C$ sucrose) i s placed i n the f r o n t vessel ; i n f igure 6, the denser mater ia l i s symbolized by grea te r densi ty of do ts . An exact ly equal volume of the material t h a t w i l l cons t i tu te t he l i g h t end of t h e gradient (e&. , le sucrose) i s placed i n the r ea r vesse l . combined volume of t he two solut ions i s chosen t o j u s t equal the volume of one centrifuge tube. vessels is then removed, and t h e magnetic s t i r r e r s a r e s t a r t e d and adjusted t o produce thorough mixing of t he solut ion i n the f ron t vesse l . Then the clamp on the o u t l e t tubing from the f ron t vesse l is loosened, and the gradient solut ion is allowed t o flow slowly down the s ide of the centrifuge tube. Every m l of solut ion removed from the f ront vesse l i n to the centrifuge tube r e su l t s i n one-half m l of solut ion flowing from the r ea r vesse l i n to the f ront vesse l . The solut ion i n the r ea r vesse l i s of low density, and this low-density solut ion i s mixed w i t h t he high- densi ty solut ion i n the f r o n t vessel , s o t h a t densi ty of the solut ion i n the f ron t vesse l gradually decreases as solut ion flows from it i n t o the centrifuge tube. The r e s u l t i s a solut ion of gradually decreasing densi ty from the bottom of t h e centr i fuge tube t o the top (as symbolized i n f igures 5 and 6) . temperature f luc tua t ions , such gradients a re s tab le f o r many hours. In general, however, it is w i s e not t o prepare densi ty gradients u n t i l shor t ly before they a r e t o be used.

Figure 6 shuws a simple, homemade apparatus t h a t

The

The clamp on the tubing connecting the two

If not disturbed o r exposed t o d r a f t s o r large

The second general category of densi ty gradient centr i fugat ion is isopycnic--zonal or simply isopycnic centr i fugat ion (Meselson e t al., 1957). This category of densi ty gradient centr i fugat ion i s a l s o some- times referred t o as "sedimentation equilibrium i n a buoyant densi ty gradient" or as "band centr i fugat ion i n CsC1" (Schumaker, 1967). general fea tures of t h i s method a r e described i n t ab le 7 and a r e i l l u s t r a t e d i n f igure 7. sample layered on it ( r i g h t tube i n f igure 7) as i n zonal centr i fugat ion, or sample and gradient mater ia l may be uniformly dispersed throughout the tube ( l e f t s ide of f igure 7) before s t a r t i n g the run. In e i t h e r instance, centr i fugat ion continues f o r a very long period of time, and t h e densi ty gradient formed i n t h e tube represents an equilibrium between sedimentation of the so lu te forming the gradient and diff'usion of t h i s so lu te against t he cent r i fuga l f i e l d . Hence, the f i n a l density gradient i n isopycnic centr i fugat ion i s a function of speed of the centr i fuge and s i z e of the

The

The gradient may e i t h e r be preformed and the

273

Fig. 6. Schematic diagram of an apparatus fo r preparing l i nea r density gradients. l eve l with the solutions chosen t o make the gradient. The solution chosen f o r the dense end of the gradient i s placed i n the f ront vessel (greater densi ty of dots) , and the solution chosen f o r the l i g h t end of the gradient i s placed in the rear vessel (fewer dots). When the tube connecting the vessels is opened and the ou t l e t tube i s opened, a l i nea r densi ty gradient such as t h a t shown i n the tubes i s produced.

Two vessels of equal diameters a re f i l l e d t o the same

274

A

H

C 6

Fig. 7. A diagrammatic representation showing separation of pa r t i c l e s by isopycnic-zonal densi ty gradient centr i fugat ion. beginning of centrifhgation, the p a r t i c l e s t o be separated may be d i s t r ibu ted uniformly i n a homogeneous suspending medium (A) or t h e p a r t i c l e s may be layered i n a t h i n zone on a preformed gradient (B) as i n rate-zonal dens i ty gradient centr i fugat ion. the f i n a l densi ty gradient is formed by the c e n t r i m a l f i e l d i t s e l f (of course, t h e change involved i n formation of the gradient is much grea te r f o r A than f o r B ) and separation of the p a r t i c l e s depends so le ly on the differences i n their dens i t ies (note that the l a rge r p a r t i c l e s a r e banded higher in the gradient ( C ) than t h e smaller p a r t i c l e s ) . From Anderson (1966) and reproduced by persmission of the author and the U .S. Department of Health, Education and Welfare.

A t the

I n e i t h e r s i t ua t ion ,

275

gradient-forming so lu te and i s formed during the run i t s e l f . preformed gradient is used, t he shape of t h i s gradient may change during the run depending on speed of t he centrifuge, e t c . gation m s a r e continued u n t i l each subcel lular component has sedimented and banded in t h e gradient a t a point exact ly equal t o i t s densi ty (center tube, f igure 7 ) . centr i fugat ion and zonal centrifugation, isopycnic cent r i fuga l separations a r e based so le ly on densi ty of t he components, and s i z e and shape have no e f f ec t on isopycnic cent r i fuga l separations (note the l a rge r p a r t i c l e s banded higher i n the schematic diagram i n center tube, f igure 7) . Because width of sample bands decreases with increasing ro to r speed, and because high r o t o r speeds move Components t o t h e i r dens i t ies more quickly, isopycnic centr i fugat ion runs are normally done a t the highest possible speeds. This combination of high speed and long runs places severe demands on cent r i fuga l e q u i p e n t , but very high resolut ions a re possible a t t he very grea t cen t r i fuga l forces used. For example, one of t he c l a s s i c a l separations done by isopycnic centr i fugat ion involved resolut ion of s ing le s t rands of DNA t h a t d i f fe red only i n t h a t one of the s t rands contained a heav isotope of nitrogen, l S N , whereas the

nitrogen. (approximately 1.710 and 1.724 g/cm3), and yet they were completely separated (Meselson and Stah l , 19%).

centr i fugat ion (Hearst and Schmid, 1973).

Even i f a

Isopycnic cent r i fu-

Consequently, i n contrast t o d i f f e r e n t i a l

other s t rand contained only 14 N, t he na tura l ly occurring isotope of Density of these two DNA strands d i f fe red by only 0.014 g/cm3

Tlnder favorable conditions, densi ty differences of only 0.0005 g/cm 3 can be resolved by isopycnic

It is obvious, of course, t h a t t he mater ia l used t o form the densi ty gradient i n isopycnic centr i fugat ion must be dense enough t o "f loat" a l l subcel lular components i n t he sample. On the other hand, t he dens i ty gradient i n zonal centr i fugat ion i s used only t o s t a b i l i z e the sedimenting zones, and there i s no requirement that the gradient be as dense or denser than the sedimenting components i n zonal centr i fugat ion. These differences i n requirements obviously a f f e c t t h e choice of mater ia l used t o form the gradient i n densi ty gradient centr i fugat ion. A s shown i n t ab le 8, sucrose and Fico l l , the two most popular gradient mater ia ls f o r zonal centr i fugat ion do not form extremely dense solut ions, and these mater ia ls are , therefore, not as usefu l f o r isopycnic centr i fugat ion as they a r e f o r zonal centr i fugat ion. and epichlorohydrin, and because it forms solut ions with low osmolarity, it i s frequently used i n densi ty gradient centr i fugat ion s tudies involving i n t a c t c e l l s where high concentrations of sucrose or s a l t s would cause i r r eve r s ib l e damage t o the c e l l . Although cesium chloride forms very dense solut ions and would, therefore , seem t o be the mater ia l of choice f o r forming densi ty gradients, it is extremely expensive ($100-400 of C s C l would be required t o f i l l six, 25-1 centrifuge tubes) and i r r e - vers ib ly destroys many subcel lular organelles a t t he high concentrations (up t o 7 M ) used Fn densi ty gradients . Metrizamide (2-(3 acetamido - 5-N- methylacetamide -2,4,6 -triodobenzamido ) -2-deoxy-D -glucose ) has recent ly been introduced as a densi ty gradient mater ia l . It i s capable of forming f a i r l y dense solut ions a t lower osmolarit ies than sucrose and may be usefu l f o r some appl icat ions involving densi ty gradient centr i fugat ion of proteins or ribosomes. A t present, however, Metrizamide is a l s o qui te expensive.

F i c o l l is a copolymer of sucrose

TABLE 8. MATERIAIS USEFUL FOR FORMING DENSITY GRADIIENTS

Approximate Maximum maximum Approximate densi ty viscosi ty maximum a t 5 O c a t 5' c c once n t r a t ion

Material (g/cm3 1 (Centipoise) (mg/mJJ

Sucrose 1.32 620 860

F ico l l 1.16 1020 465

Cesium chloride 1.85 --- 1160

510 Potassium bromide 1.32 --- 960 Potassium c i t r a t e 1 .51 --- 950 Potassium t a r t r a t e 1.48 ---

Metrizamide 1.47 246 850

Densities of Cells and Subcellular Components

Densities of c e l l s and some subcellular components as determined by isopycnic centrifugation a re given i n tab le 9. material i n which the density determination was made a f f ec t s the hydration and, therefore, the density of the component, the gradient material used f o r the density determination i n table 9 is given i n parentheses after each density. It is evident from comparing component dens i t ies i n t a b l e 9 with properties of the gradient materials i n t ab le 8 t h a t C s C l would be necessary f o r isopycnic s tudies of glycogen, DNA, and RNA. A l t h o u g h it would be possible t o use sucrose i n isopycnic s tudies of proteins and nuclei , such s tudies would be extremely d i f f i c u l t because of t he exceedingly high v iscos i t ies of the very concentrated sucrose solutions necessary f o r such s tudies . Hence, in pract ice , t he use of sucrose in isopycnic s tudies i s l imited t o cmponents w i t h t h e densi ty of lysosomes or l e s s . Because they are useful at opposite ends of the densi ty range i n t ab le 9, sucrose and C s C l have been by far the most widely used materials i n density gradient centrifugation, and approximately 95% of all densi ty gradient s tudies done use cneof these two materials t o form the gradient,

Because the gradient

TABU 9. DENSITIES OF SOME SUBCELIULAR CONSTITUENTS~

Constituent Approximte density (g/cm3)

Whole c e l l s

Microsomes

T-tubules and sarcolemma

Sarcoplasmic r e t i c u l a r membranes

Mitochondria

Lys os ones

Muscle lysosomes

Protein

Nuclei

Ribosomes

Glycogen

DNA

RNA

1.06 - 1.12 (F ico l l )

1.12 - 1.18 (sucrose)

1.10 - 1.12 (sucrose)

1.13 - 1.17 (sucrose)

1.14 - 1.19 (sucrose)

1.15 - 1.20 (sucrose)

1.18 (sucrose)

1.28 - 1.33 (sucrose)

1.29 - 1.35 (sucrose)

1.43 (sucrose) t o 1.54 ( C s C 1 )

1.61 - 1.65 (csci) 1.69 - 1-75 ( C s C 1 )

1.85 - 1-95 ( C s C 1 )

a Gradient mater ia l used t o determine the density of each component is given i n parentheses imnediately a f t e r each density. I n many instances, the type of gradient mater ia l used w i l l a f f ec t the hydration and, therefore, the density of the pa r t i c l e . Some dens i t ies were determined a t temperatures above 25OC t o permit grea te r solute so lub i l i t y and, therefore, greater density (cf c . C s C l density f o r RNA and i n tab le 8) .

PURIFICAlTON OF SUBCELTULAR COMPONEXTS OF MUSCLE BY DENSITY GRADISN'T CENTRIFUGATION

Application of Density Gradient Centrifugation t o Crude Muscle Ce l l Homogenates

The remainder of t h i s review w i l l discuss three d i f f e ren t examples of densi ty gradient pur i f ica t ion of subcel lular components. example (f igure 8 ) is an attempt t o pur i fy subcel lular components d i r e c t l y frcgn crude muscle c e l l homogenates by using zonal centr i fugat ion. Because crude muscle homogenates contain a l l t h e subcel lular components of muscle c e l l s i n one suspension, the quant i ty of any s ingle component i n the homogenate is necessar i ly l imited. Consequently, large volumes of the homogenate a r e required t o obtain appreciable quant i t ies of any s ingle pur i f ied subcel lular component and such large volumes require that a zonal r o t o r be used. with 1500 m l capacity w a s used; 60 m l of a muscle homogenate made by homogenizing f rog s k e l e t a l muscle i n three volumes (v/w) of 0.25 M sucrose, 1.8 mM CaC12, 1 mM T r i s buffer a t pH 8 f o r th ree minutes i n a Waring Blender was applied t o the gradient . Only four c l ea r ly defined f rac t ions could be obtained by zonal centrLfugation of t he crude muscle homogenate under these conditions (figure 8 ) even though it is possible t o resolve crude muscle hmogenates in to f i v e p a r t l y pur i f ied f rac t ions by using only d i f f e r e n t i a l centr i fugat ion ( t ab le 5 ) . Furthermore, biochemical and electron microscope analyses of t h e four f rac t ions obtained by zonal centr i fugat ion showed that a l l four f rac t ions were mixtures of severa l components. soluble proteins , nucleic acids, and other soluble mater ia l as wel l a s smaller pa r t i cu la t e mater ia l (probably the microsomal f r ac t ion i n t a b l e 5 ) . The f r ac t ion labeled "2" contained la rger pa r t i cu la t e mater ia l (probably t h e heavy microsomal f r ac t ion of t a b l e 5), some mitochondrial fragments, and some small f i laments . Fraction 3 contained collagen f i b e r s and myofibrils that appeared free from sarcoplasmic r e t i c u l a r membranes, whereas f r ac t ion 4 contained collagen f i b e r s and myofibrils t h a t had an abundant amount of sarcoplasmic r e t i c u l a r membranes adsorbed t o t h e i r surface. any s ingle f r ac t ion . It is clear , therefore , that zonal centrifugation, when used d i r e c t l y on crude muscle cellhomogenates, does not s a t i s f a c t o r i l y resolve the subcel lular components present i n such homogenates. t he re a r e severa l reasons f o r t h e ineffectivgless D f zonal centr i fugat ion when applied t o whole c e l l hmogenates, one of t h e pr inc ipa l sources of d i f f i c u l t y i s the presence of cmponents with such very widely d i f f e r h g sedimentation coef f ic ien ts i n t h e sample t h a t it i s impossible t o choose a centr i fuge speed and time that w i l l not completely sediment t h e heaviest components but w i l l a l so sediment t he lighter components a t l e a s t a small distance i n t o the gradient (note t h a t f r ac t ion 1 i n f igure 8 i s s t i l l on t h e surface of t h e gradient)

The first

I n the experjnent shown in f igure 8, a ro tor

The f r ac t ion labeled "1" contained

Mitochondria and nuclei were not c l ea r ly resolved i n t o

Although

279

io

9

W In 0

3 In

30 5

0 1 4 8 12 46 20 24

FRACTION NUMBER (40 ml each)

Fig. 8. Separation obtained by zonal density gradient centr i fugat ion of a crude hmogenate of f rog s k e l e t a l muscle. crude muscle homogenate i n 250 mM sucrose, 1.8 mM CaC12, 1 mM T r i s o H C 1 , pH 8, w a s layered onto a sucrose gradient made by layer ing 200 m l of 13% sucrose over a 700 ml continuous, l i nea r 33 t o 54$ sucrose gradient with an underlay of 300 ml of 5576 sucrose. overlay of 140 m l of 1.8 I13M CaC%, 1 mM T r i s O H C l , pH 8, w a s i n t r o - duced over t h e sample. Loading w a s done i n a zonal ro to r a t 1000 rpm, the ro to r was accelerated t o 2000 rpm f o r 10 min, then decelerated t o 1000 rpm, and immediately unloaded by pumping 5576 sucrose i n t o the outer edge of the r o t o r . The e f f luent steam w a s monitored a t 260 nm ( s o l i d l i n e ) and col lected i n 40 m l f r ac t ions . Sucrose concentration of each f r ac t ion was measured refractometr ical ly (dotted l i n e ) . pended f o r e lectron microscope and biochemical assays. from Barber and Canning (1966) by permission of the authors and the U.S. Department of Health, Education and Welfare.

A t o t a l of 60 m l of

An

Aliquots of each f r ac t ion were pel le ted and resus- Reproduced

280

Fractionation of the Muscle Microsomal Fraction by Density Gradient Centrifugation

The data in f igure 8 demonstrate that zonal centrifugation cannot be used e f fec t ive ly t o separate all subcellular components from a crude muscle c e l l homogenate. Figures 9, 10, U, and I2 show the r e su l t s when density gradient centrifugation i s used t o purify f ract ions t h a t have been separated and pa r t ly purif ied by d i f f e r e n t i a l centrifugation. A microsomal f rac t ion (see t ab le 5 ) was obtained by d i f f e r e n t i a l centrifugation between 15,000 x 8 f o r 20 minutes and 123,000 x & for 57 minutes of a crude homogenate of rabbi t ske l e t a l muscle (Heuson- Stiennon e t al., 1972), This microsomal f rac t ion was resuspended i n 0.25 M sucrose, 0.05 M KC1, and the suspension was layered onto a 0.74 M t o 2.05 M continuous l i nea r sucrose gradient. The subcellular components in t h e microsomal suspension were separated by isopycnic density gradient Centrifugation (f igures 9 and 10) i n t o three fract ions: (1) a low-density f rac t ion (density 1.10-1.12 labeled 1 i n figure 9 and 10); (2) a high-density f rac t ion (density 1.13-1.17 labeled 2 i n f igures 9 and 10); and (3) a contaminating fract ion (labeled C i n figure 9 and unlabeled peak j u s t before l e t t e r b i n f igure 10). Biochemical analyses showed t h a t the low-density f rac t ion had no Ca2+-seque t e r ing a b i l i t y , but t h a t the high-density f rac t ion possessed potent Ca'+-binding a b i l i t y . Neither the low-density nor the high-density f ract ions had any cytochrome oxidase ac t iv i ty , so both f rac t ions were therefore probably f r ee from contamination by mitochondrial membranes . "he contaminating fract ion contained nearly a l l cytochrome oxidase a c t i v i t y present in the or ig ina l pa r t ly purif ied microsomal f ract ion, so t h i s f rac t ion probably represented the mitochondrial contamination present i n the or ig ina l microsomes. Electron microscope observ&tions (figures 11 and 12) showed t h a t the low-density f rac t ion was heterogeneous and contained many tubular or tadpole-like s t ructures ( f igure 11) t h a t resembled the T-tubles seen i n sections of i n t ac t muscle (cf. f igure 2 and No. 12-16 i n f igure 11) as wel l a s fragments of the outer c e l l membrane (sarcolemmal fragments, No. 17 i n figure 11) that had been ruptured when t h e t i s sue had been passed through the meat grinder (see paper by D r . Matsushima l a t e r in th is volume). Indeed, in a few instances (no. 14 i n f igure ll), what appeared t o be a T-tubule s t i l l lying between two l a t e r a l c is ternae could be seen in sections of t he low-density f rac t ion . f ract ion, on the other hand, was s t ruc tu ra l ly homogeneous and consisted of vesicular s t ructures 90-120 nm i n diameter i n e i the r negatively stained (no. 18 in f igure 12) or posi t ively stained (numbers 19 and 20 i n f igure 12) sections. a re pieces of t h e sarcoplasmic r e t i c u l a r membrane (see f igure 2 ) that have been ruptured by grinding and homogenization. in both the low-density and high-density f ract ions a r e approximately 7.5-10.0 nm, and i n some instances (no. 20 i n f igure I2), a t r i p l e - layered or subunit s t ruc ture of t he membrane can be discerned. D r . Stromer w i l l discuss s t ructure of t he un i t membrane in d e t a i l l a t e r i n t h i s volume. low-density f rac t ion does not, these electron microscope observations indicate that a l l Ca2+ - accumulating a b i l i t y in s k e l e t a l muscle i s i n

The high-density

It i s very likely that these vesicular s t ructures

Membrane thicknesses

Because the high-density f ract ion accumulates Ca2+ but the

28 1

Fig. 9. Subfractions of a muscle microsomal f r ac t ion are seen as light-colored bands i n t h i s centrifuge tube after centr i fugat ion a t 25,000 rpm f o r 15 hrs. Rabbit l eg muscle was ground, suspended i n 180 mM sucrose, 100 mM phosphate buffer , pH 7.2, by using a Pot te r homogenizer f o r 3 min, and sedimented a t 1500 x B;. f o r 5 min t o remove nuclei and myofibrils . K C 1 was added t o t h e supernatant t o give a final KC1 molarity of 250 mM ( t o keep myofibr i l lar proteins soluble), and a microsomal p e l l e t was sedimented between 15,000 x and U 3 , O O O x B;. f o r 57 min. The microsomal p e l l e t was resuspended i n 250 mM sucrose, 50 mM K C 1 and centrifuged a t low speed t o remove contaminating myofibr i l lar protein. The resu l t ing supernatant w a s layered onto a continuous l i n e a r sucrose densi ty gradient (25 t o 7046 sucrose) and centrifuged a t 25,000 rpm f o r 1 5 hours t o produce the separation seen i n th is tube. f i gu re ) i s banded a t a densi ty of 1.10-1.12 and subfraction 2 i s banded a t a densi ty of 1.13-1.17. C=contanbating band, arrow indicates t op of t h e gradient. and reproduced by permission of t he Rockefeller University Press and the authors.

f o r 20 min

Subfraction 1 (area labeled 1 i n

From Heuson-Stiennon L- e t a l . (1972)

282

MOLES SUCROSE

1.e

1.6

l , L

12

10

O B

06

0.L

02

Fig. 10. The d i s t r ibu t ion of proteins ( so l id l i n e labeled "a") and o p t i c a l dens i t i e s (dashed l i n e labeled '%") measured a t 280 nm of a muscle, microsomal f r ac t ion a f t e r density gradient centr i fugat ion a t 39,000 rpn f o r 2 h r is shown i n t h i s f igure . somal f r ac t ion was prepared as described i n the legend t o Fig. 9 . A continuous, l i n e a r sucrose densi ty gradient from 25 t o 704& sucrose was used. The f rac t ions Labeled 1 and 2 correspond t o those s imi la r ly labeled i n Fig. 9 . concentration of the sucrose gradient . From Heuson-Stiennon e t a l . (1972) and reproduced by permission of t he Rockefeller University Press and t h e authors.

The muscle micro-

The dotted l i n e labeled "ctr i s t h e m o l a r

f.

Fig. 11. These electron micrographs of the low densi ty f r ac t ion Labeled i n Fig. 9 and 10 show that t h i s f r ac t ion contains numerous tubular, t a i l - l i k e s t ruc tures (arrows i n a a n d severa l t r i a d s (T i n 13) t h a t a r e reminiscent o t t he t r i a d s seen i n sectioned muscle. The area Labeled T i n 13 i s shown a t higher magnification i n 14 where i t s resemblance t o the t r i a d i s qui te s t r ik ing . Higher magnifications of the fxbular s t ruc tures observed i n I 2 a r e shown i n 15 and 16; these s t ruc tures may be segments of T-tubules broken during grinding and homogenization. “he typ ica l , folded, sarcolemmal membranes seen i n the low-density f r ac t ion a r e shown i n 17; t h e granular mater ia l (arrows ) may represent fragmented, f ibrous proteins . The sarcolemma i n t h i s f r ac t ion has been broken during grinding and homogenization because if t he sarco lemal sheath had remained in t ac t , it would have sedimented with the w o f i b r i l l a r f r ac t ion . 12, 13 and 17; X25,900. 14, 15 and 16; XlO5,OOO. reproduced by permission of t he Rockefeller University Press and the authors.

FromHeuson-Stiennon e t a l . (1972) and

284

Fig. 12. These electron micrographs of the high-density f rac t ion labeled 2 in F i g . 9 and 10 show that this f rac t ion is s t ruc tu ra l ly more homogeneous than the law density f ract ion, and consists almost en t i r e ly of thin-walled ves ic les . a posi t ively stained section is shown i n 19. A t the higher mgnif icat ions shown i n 19, the pos i t ive ly stained membranes appear t r iple- layered (arrows), and minute spots are sometimes observed. The ves ic les i n t h i s f rac t ion a re probably formed by resealing of sarcoplasmic r e t i cu la r tubules that a re ruptured during grinding and homogenization. 18 and 19; X25,OOO; 20; X137,OOO. and reproduced by permission of the Rockefeller University Press and the authors.

A negatively stained view is shown i n 18 and

FromHeuson-Stiennon e t a l . (1972)

the sarcoplasmic r e t i c u l a r membranes and that the T-tubules, l a t e r a l c is ternae, and sarcolemmal membranes of s k e l e t a l muscle have no Ca2+ - accumulating a b i l i t y . It i s evident from t h i s b r i e f s w r y of the evidence i n f igures 9, 10, 11, and 12 t h a t carefu l densi ty gradient pur i f ica t ion of p a r t l y pur i f ied subcel lular crmponents obtained by d i f f e r e n t i a l centr i fugat ion can r e s u l t in precise loca l iza t ion of biochemical functions. The microsomal pur i f ica t ion summarized i n f igures 9-12 a l s o i l l u s t r a t e s the usefulness of densi ty gradient centr i fugat ion t o purify pa r t ly pur i f ied f rac t ions obtained by d i f f e r e n t i a l cen t r i fu- gation i n contrast w i t h t he i n a b i l i t y of densi ty gradient centr i fugat ion t o purify subcel lular components from crude muscle c e l l homogenates ( f igure 8 ) .

Pur i f ica t ion of Muscle Polysomes by Density Gradient C e n t r i m a t i o n

The l a s t example of the use of densi ty gradient centr i fugat ion t o pur i fy subcel lular components from s k e l e t a l muscle involves zonal cen t r i fuga l pur i f ica t ion of the polysomes t h a t t r a n s l a t e f o r synthesis of myosin ( f igu re 13 ) . chicken s k e l e t a l muscle myoblasts a r e prepared by asp i ra t ing t h e c e l l s gently i n 250 mM KC1, 10 mM MgC12, 10 mM T r i s HC1, pH 7.4, 0.5$ Tri ton X-100. The lysed c e l l s are centrifuged f o r 10 minutes a t 12,000

and the supernatant is then layered onto a continuous l i nea r 1.5 t o t8 sucrose gradient . This concentration of sucrose i s obviously not su f f i c i en t t o " f loa t" polysomes (see tab les 8 and 9 ) so the separation i s rate-zonal. Centrifugation a t l50,OOO x 13 f o r 90 minutes separates the 12,000 x g supernatant in to a range of d i f f e ren t f rac t ions ( f igu re 13) . not sediment very f a r and probably represent p r o t e b s and other soluble, u l t r a v i o l e t -absorbing substances i n the 12,000 x g supernatant that was layered onto the gradient . chains, the m-RNA t h a t codes f o r synthesis of myosir, must a l s o be la rge , and polysomes ( i .e . , m-RNA plus ribosomes) that t r a n s l a t e f o r myosin would be expected t o have sedimented a considerable distance in to the gradient . Assay of f rac t ions obtained from the densi ty gradient indicated t h a t only f rac t ions 3-6 were ab le t o catalyze synthesis of t h e heavy chains Df myosin ( f igure 13) . material i n these f rac t ions indicated ( f igure 14 ) t ha t it contained very large polysomes similar t o those described by Heywood e t a l . (1967) as capable of supporting myosin synthesis . Consequently, it i s possible, by using a few d i f f e r e n t i a l cen t r i fuga l s teps followed by density gradient centrifugation, t o i s o l a t e from a crude muscle homogenate a c l a s s of polysomes that t r a n s l a t e f o r synthesis of a s ingle protein.

Homogenates of 76-hour c e l l cul tures of fused

The f rac t ions on t h e right s ide i n f igure 13 a re those that did

Because myosin contains very large polypeptide

Electron microscope examination of the

CONCLUSIONS

Although t h i s review has been r e s t r i c t e d t o a discussion of t he use of centr i fugat ion t o separate subcel lular components and other p a r t i c l e s from each other, t he universal applicability and unique

286

1 . 6

1 . 4

1 . 2

I E 1 . 0 0 Io N v

w 0 z -2 0.e CL 0 VI

4 m

0.6

0 . 4

0 . 2

0

zoo0 I - Ln

E

V a

I 5 0 0 g - - 4 I V

> >

1 0 0 0 f z v)

0 > x

w

500 z v) b- z 3

0 u

0

40% S U C R O S E F R A C T I O N N U M B E R 1 5 % S U C R O S E

Fig. 13. Density gradient p ro f i l e of a polysame f rac t ion prepared from a homogenate of 76-hr cul ture of 'fused chick ske le t a l myoblasts. Fused chick s k e l e t a l myoblasts were lysed by asp i ra t ing gently i n 250 mM KC1, 10 1~24 MgC12, 10 mM Tris.HC1, pH 7.4, 0.546 Triton X-100, and t h i s homogenate was centrifuged f o r 10 min at 12,000 x g t o remove myofibrils, unbroken c e l l s , nuclei, and mitochondria. supernatant from t h i s centrif'ugation i s layered onto a continuous, l inear 15 t o 40$ sucrose density gradient and centrifuged f o r 90 min a t 150,000 x g. solution i n t o the bottom of t h e tube and monitoring the e f f luent a t 260 nm (heavy continous l i n e ) . ApproxFmately two ml f rac t ions were col lected. Polysomes i n each f rac t ion were pel le ted by centrifuging a t l50,OOO x f o r 2 hrs through 1.5 M sucrose and were then assayed f o r a b i l i t y t o incorporate 3H-leucine in to the heavy chain of myosin ( l i n e connected by do t s ) . rad ioac t iv i ty incorporated in to t h e 200,000 molecular weight band isolated by SDS-polyacrylamide g e l electrophoresis of the incorporation mixture a

The

The tubes a re unloaded by punping dense sucrose

Incorporation w a s measured as amount of

- ' A *

Fig. 14. This electron micrograN of the heavy polysome fractions isolated from a 15-40q& l inear , continous sucrose density-gradient centrifugation of the polysome fract ion prepared from chick ske le ta l myoblasts (see Fig. 13) shows tha t these heavy polysome fractions contain polysomes large enough t o be responsible fo r synthesis of %he heavy chain of myosin. i n the polysome shown i n the upper center of t h i s micrograph ( c f . Heywood e t al., 1967)

F i f t y t o s ix ty ribosomes may be counted -

~86,200.

competency of the centr i fuge t o pur i fy subcel lular p a r t i c l e s should be evident from the f e w examples given here . Muscle b io log i s t s and meat s c i e n t i s t s have, i n general, not used densi ty gradient pur i f ica t ion as widely as many other molecular and c e l l u l a r b io logis t s , and s igni f icant advances i n our understanding of the muscle c e l l and of meat qua l i ty may be possible by carefu l application of density gradient pur i f ica t ion t o some of t he problems of muscle biology and meat science. Hopefully, t h i s review m i g h t prompt such appl icat ions.

ACKNOW l X D G m T S

We a r e g ra t e fu l t o Darlene Markley f o r drawing f igures 4 and 6 and t o Janet Stephenson f o r ass is tance with the t i s s u e cu l ture s tud ies . The patience and assis tance of Joan Andersen and Barbara Hallman i n draf t ing t h i s manuscript is also gra t e fu l ly acknowledged.

LITERATURE: CITED

Abeloff, M . D . , R . J . Mangi, T . G . Pretlow and M . R . Mardiney. 1970. I so l a t ion of human leukemic b l a s t s from peripheral blood by densi ty gradient centr i fugat ion. J . Lab. Clin . Med . 75 : 703.

Anderson, N . G . 1965. Zonal u l t racent r i fbga t ion . & Fractions, No. 1, Beckman Instruments, Inc., Palo Alto, California, p . 2 - 8 .

Anderson, N . G . 1966. The Development of Zonal Centrifuges. N a t l . Cancer I n s t i t u t e Monograph 21. Office, Washington, D .C . pp . 1-526. U .S . Government Pr int ing

20402.

Anderson, N . G . 1968. Analytical techniques f o r c e l l f rac t ions . V I I I . Analytical d i f f e r e n t i a l centr i fugat ion i n angle-head ro to r s . Biochem . 23 : 72 . A n a l .

Barber, M . L, S. and R . E . Canning. 1966. Extraction of con t r ac t i l e proteins from myofibrils prepared by rate-zonal centr i fugat ion. The Development of Zonal Centrifuges and Ancillary Systems f o r Tissue Fractionation and Analysis. Cancer I n s t i t u t e Monograph 21, U.S. Department Health, Education and Welfare. p . 345-360.

- I n

( N .G . Anderson, ed . ), National

Boone, C . W . , G . S . Hare11 and H. E . Bond. 1968. The resolut ion of mixtures of viable m a m l i a n c e l l s i n t o homogeneous f rac t ions by zonal centr i fugat ion. J. Cel l Biol. 36:369.

Brakke, M. K. 1951. Density gradient centr i fugat ion: a new separation technique. J. Am. Chem. SOC. 73:1847.

Bra-, M. K. 1953. Zonal separations by density-gradient centr i fugat ion. Arch. Biochem. Biophys . 45:275.

Brentani, R . , M . Brentani and I. Raw. 1967. Zone sedimentatim analysis of RNA i n angle-head ro to r s . Anal. Biochem. 20:361.

Charlwood, P . A . 1963. Applications of radioact ively labeled marker proteins i n density gradient u l t racent r i fuga t ion . 5:226.

Anal. Biochem.

deDuve, C . and J. Berthet. 1954. i n t h e study of t i s s u e enzymes. I n t . Rev. Cytol . 3:225.

The use of d i f f e r e n t i a l centr i fugat ion

deDuve, C., J. Berthet and H . Beaufay. 1959. Gradient centr i fugat ion Theory and appl icat ions. of c e l l p a r t i c l e s .

Chem. 9:326. Prog . Biophys . Biophys .

Fisher, W . D. , G. B. Cline and N . G . Anderson. 1964. Density gradient centr i fugat ion i n angle-head ro to r s . Anal. Biochem. 9:477.

Gauthier, G . F. 1970. The u l t r a s t ruc tu re of three f i b e r types i n mammalian s k e l e t a l muscle. I n The Physiology and Biochemistry of Muscle as a Food, 2. Marsh, ea . ) . (E . J .Br i skey , R . G . Cassens and B . B.

The University of Wisconsin Press, Madison. p . 103-130.

Hearst, John E . and Carl W . Schmid. 1973. Density gradient sedimentation equilibrium. S . N . Timasheff, ed . ) . & Methods i n Enzymoloa, Vol . XNII . ( C .H .W . H i r s and

Academic Press, Inc . , New York. p. lll-127.

Heuson-Stiennon, Jeanine-Anne, Jean-Claude Wanson and P. Drochmans . 1972. I so l a t ion and character izat ion of the sarcoplasmic reticulum of s k e l e t a l muscle. J. Ce l l Biol. 55:471.

Heywood, S. M. , R . M. Dowben and A . Rich. 1967. The iden t i f i ca t ion of polyribosomes synthesizing myosin. Proc. N a t l . Acad. Sc i . 57:1002.

Martin, R . G. and B. N . Ames. 1961, A method f o r determining the sedimentation behavior of enzymes : appl icat ion t o protein mixtures. J. Biol. Chem. 236:1372.

Meselson, M . and F. W . S tah l . Escherichia - c o l i .

1958. The rep l ica t ion of DNA in Proc . N a t l . Acad . Sc i . 44 :671.

Meselson, M., F. W . S t a h l and J. Vinograd. 1957. Equilibrium sedimentation of macro-molecules i n densi ty gradients . Proc. Natl. Acad. Sc i . 43:581.

Mil ler , H. C . and G. Cudkowicz. 1971. Density gradient separation of marrow c e l l s r e s t r i c t e d f o r antibody c l a s s . Science 171:913.

Peachey, L. D. 1970. Form of the sarcoplasmic reticulum and T-system of s t r i a t e d muscle. a Food, 2. University of Wisconsin Press, Madison.

I n The Physiology and Biochemistry of Muscle as

p. 273. (E . J. Brgkey, R. G . Cassens and B. B. Marsh, ea.). The

290

Pickels, E . G. 1943. Sedimentation i n the angle c e n t r i m e . J. Gen. Physiol . 26 : 341.

Pretlow, T. G . 1971. Estimation of experimental conditions t h a t permit c e l l separations by ve loc i ty sedimentation on i sokine t ic gradients of F i c o l l i n t i s s u e cul ture medium. Anal. Biochem. 41:248.

Pretlow, T . G . and C . W. Boone. 1968. Centrifugation of mammalian c e l l s on gradients: a new ro to r . Science 161:911.

Pretlow, T. G. and C . W . Boone. 1969. Separation of mammalian c e l l s using programmed gradient sedimentation. Exptl . Mol. Path. 11:139.

Pretlow, T . G. and C . W . Boone. 1970. Separation of malignant c e l l s from transplantable rodent tumars . Exptl . Mol. Path. 12:249.

Pretlow, T . G . and I . M . Cassady. 1970. Separation of mast c e l l s i n successive s tages of d i f f e ren t i a t ion using programmed gradient sedimentation. Am. J . Pa th . 61:323.

Schumaker, V . N. 1967. Zone centr i fugat ion. Adv. Biol. Med. F'hys. 11:245.

Schumaker, V . and A . Rees. 1969. Theory of d i f f e r e n t i a l centr i fugat ion i n angle-head ro to r s . Anal. Biochem. 31:279.

M . D . Judge: We thought it w m l d be appropriate t o c a l l the speakers back one a t a time now and l e t them "top o f f " t h e i r p re sen ta t ims . Spec i f ica l ly we asked them t o make a comment about a way of applying the research method discussed and then you may have questions or comments.

R . M . Robson: proteolyt ic enzyme j u s t went through

A s I was saying be fwe , w e a r e in te res ted i n t h i s and i t s e f f e c t on myofibril lar proteins . Before, I

individual ly pur i f ied proteins; however, i n some work done by Dennis Olson i n our laboratory w e ac tua l ly digested the e n t i r e myofibril w i t h this enzyme. myofibril i n SDS and then electrophoresing it. myosin heavy chains, t he alpha ac t in in , ac t in and tropomyosin. Essent ia l ly we found t h a t t h i s component, which we presume t o be troponin T i n bovine muscle, i s very quickly l o s t f r D m t he myofibril . same time one ge ts a degradation product showing up. One ge ts a somewhat s imilar occurence i f one simply ages muscle a f t e r death. aging progresses, t he band disappears.

T h i s i s a n example of put t ing the e n t i r e You can iden t i fy the

I n approximately the

A s post-mortem

C . S . Chung, is our laboratory, is studying myofibr i l lar proteins prepared from muscle that has been cooked f o r d i f f e ren t times and temperatures. This is another type of problem t h a t can be done with t h i s technique. A s you heat the muscle past about 6ooc you s t a r t t o pick up ex t ra bands. Now these a r e not degradation products; they a re , i n f a c t , sarcoplasmic proteins which a r e precipi ta ted out on t h e myofibrils during cooking and then a re i so la ted on the myofibrils .

M . L. Greaser, University of Wisconsin: Could we go back t o the second s l ide? W h a t a r e the molecular weights on the l e f t s ide of those second and t h i r d bands up from the bottom?

R . M . Robson: This i s tropomyosin a t 35,000 and th i s one probably a l k a l i 1 and perhaps troponin I a t about 25,000.

M . L. Greaser: I n other words, t he re i s no troponin C there?

R . M . Robson: Troponin C in this g e l would probably be i n t h i s region, although it does not show up very wel l .

P. B. Addis, University of Minnesota: I was in te res ted i n the s l i d e of t he heat denatured pro te in . you seem t o ge t good electrophoret ic separation of t h e heat denatured proteins; do you th ink that it might be possible t o do a species iden t i f i ca t ion on cooked meat samples o r resolve a mixture of two d i f f e ren t species?

By use of SDS electrophoresis,

R . M. Robson: It m i g h t be possible but it would be a l i t t l e d i f f i c u l t . Perhaps I could have Marion Greaser comment on troponins. This i s one of t h e myofibr i l lar proteins t h a t w i l l change s l i g h t l y i n the subunit molecular weights. For instance, d i f f e ren t muscles from the same animal ( i .e. , cardiac and s k e l e t a l muscle) w i l l d i f f e r . Marion, have you checked d i f f e ren t species t o see i f they a l s o vary between animals or between species?

292

species t h a t we have looked a t do not appear t o be much d i f f e ren t i n t h e subunit molecular weights. I th ink the re i s a l i t t l e difference i n chicken muscle, f o r instance, i n the troponin T component, but f o r horse, beef and p ig muscle I th ink a l l have approximately t h e same molecular weights fo r a 11 the myofibr i l lar compnents .

M . L. Greaser: We have not done much checking on t h i s . The few

R . M . Robson: One, of course, can ident i fy mixtures of products t h a t contain soy protein or milk protein.

C E . Allen, University of Minnesota: Rich, I th ink a l l of your Perhaps the key t o D r . gels here have been on myofibr i l lar proteins .

Addis' ques t im i s found in t h e sarcoplasmic proteins a f t e r heating. Did you f ind a prec ip i ta t ion of sarcoplasmic proteins onto your myofi- b r i l l a r proteins? Would t h i s be possible f o r ident i f ica t ion of species?

R . M . Robson: If one prepares the sarcoplasmic proteins from d i f f e ren t muscle, one does get a tremendous difference i n quanity glycolyt ic enzymes. I would guess t h a t it would be a l i t t l e d i f f i c u l t t o use t h i s bu t I th ink that it is a f r u i t f u l area t h a t someone should look a t . a r e not made up of sarcoplasmic proteins because, i f he makes a n SDS g e l of t h e ex t rac t and compares it, they l i n e up per fec t ly with these ex t ra bands on t h e myofibril .

of

M r . Chung feels that t h e bands t h a t he is ge t t ing after heating

J . J . Guenther, Oklahoma Sta te : D i d you define C A F treatment?

R . M . Robson: We a r e using a pur i f ied enzyme and it i s normally added i n most of theseoresul ts w i t h a w e i g h t t o weight r a t i o of one t o two hundred done a t 25 C f o r one hour.

J . J . Guenther: Did you indicate t h a t you could not electrophorese myos in?

R . M . Robson: It is extremely d i f f i c u l t t o electrophorese it i n the absence of a denaturing solvent , F l o r i n i and Brewer t r i e d under a number of circumstances and fai led every time unless they put i n urea, which, of course, i s another dissociat ing regent.

T . R . Dutson: By heat ing above 6OoC, there seem t o be some changes t h a t t ake place i n the myofibr i l lar protein. heat denatured and j u s t not coming on with t h e gel?

Are some of these being

R . M . Robson: I th ink the problem you refer t o i s t ha t t he same protein load was not applied t o each g e l . not having equal protein loads. If one maintains the same concentration on the ge l s one does not see any difference even i n t h e bands down i n the troponin and the l ight chain region of myosin.

I th ink it is a case of j u s t

T. R . Dutson: Then the heating of these proteins up t o these high temperatures apparently doesn' t cause enough change a t l e a s t i n t h e protein s t ruc ture t o a l t e r e lectrophoret ic patterns?

293

R . M . Robson: It i s ce r t a in ly not breaking any covalent bonds. You are, i n f a c t , ge t t i ng out i n t a c t polypeptide chains.

M . D . Judge: Let's mrve on t o t h e Qther speakers and then i f we D r . Dutson, may we have time we can come back t o D r . Robson's paper.

hear from you?

T . R . Dutson: A s a b r i e f reintroduction, I would l i k e t o r e c a l l t he l a s t micrographs I showed of electronmicroprobe x-ray ana lys i s . I th ink t h i s technique i s something t h a t meat s c i e n t i s t s or muscle b io logis t s could be working with t o a grea te r extent . The preparation seems t o be very d i f f i c u l t , but I th ink that, w i t h use of various techniques such a s c r i t i c a l point drying, t h e t i s s u e could be prepared with the elements remaining local ized f a i r l y wel l i n t he t i s s u e a s i n t he i n t a c t s t a t e . There a r e a l s o problems of analyzing t i s s u e components 3r t i s s u e elements a t the low leve ls that a r e sometimes present . I t h ink one pa r t i cu la r appl icat ion here might be i n determining ce r t a in addi t ives o r cer ta in chemicals such as metals and contaminants t h a t might be present i n the muscle t i s s u e s . Also, the electron microscope and the scanning electron microscope could be used i n analyzing systems such a s meat emulsions using freeze f rac ture techniques t o break the emulsion. probably occur a t t h e membranous u n i t around the f a t portion o f a n emulsion. It seem t o me t h a t we could u t i l i z e the microscope t o give us an idea of how t h i s s t r u c t u r a l system imparts tex ture . If w e could a l so u t i l i z e the microscope and various microscope techniques i n combination with biochemical and histochemical techniques, we could g e t an idea of the way some of these components e x i s t i n a s t r u c t u r a l re la t ionship and then possibly r e l a t e t h i s t o some of t h e t e x t u r a l propert ies of muscle.

Breakage would

P . B. Addis: You showed that the Z l i n e of the red f i b e r was thicker than t h a t of the white f i b e r . How does the intermediate f i b e r Z l i n e thickness compare with the others?

T . R . Dutson: When you look a t the u l t r a - s t ruc tu re there seems t o be considerable var ia t ion between t h e two types. The intermediate f i b e r , although when s ta ined by various enzymes seems t o be ra ther d i s t i n c t , is qui te var iab le i n t h e u l t r a - s t ruc tu re . However, t he re does seem t o be a d e f i n i t e intermediate group, the Z l i nes of which appear t o have cha rac t e r i s t i c s i n between t h e red and white types as f a r as mitochondrial content and other s t ruc tures a r e concerned.

R . L. Henrickson, Oklahoma S ta t e University: Elaborate a l i t t l e fu r the r on why the Z l i n e is wider i n one case than the o ther .

T. R. Dutson: There could be a re la t ionship w i t h t h e contraction speed; whether t he pressure on the myofibril i n the white f i b e r i s any d i f f e ren t than t h a t i n the red f i b e r I r e a l l y don't know. p o s s i b i l i t y might be t h a t t he re i s more of the amorphis mater ia l i n t he Z l i n e surrounding the heavier s t r u c t u r a l proteins i n t he red f i b e r s .

Another

294

R . G . Kauffman, University of Wisconsin: I would l i k e t o have you c l a r i f y a point regarding your a b i l i t y t o prepare materials i n a completely relaxed form. you described i n a biopsy s i tua t ion i n which you d id not remove the t i s s u e u n t i l a f t e r it was fixed?

Have you ever attempted t o use the technique

T . R . Dutson: We have taken some biopsies and, of course, as soon a s we cut i n t o the muscle we saw a tremendous amount of cmt rac t ion . Our techniques could be used by biopsy but it would r e s u l t i n t he necessity t o s a c r i f i c e the animal because of t he r a the r la rge sample s i z e . relaxed sample. It is a res t ra ined sample. A relaxed sample would be something d i f f e r e n t .

I don't believe you could c a l l t he muscle a s I prepare it a

F. C . Parr ish, IDwa S ta t e University: Can you or have you looked a t changes occurring i n muscle f i b e r s during postmortem aging?

T . R . Dutson: We have looked a t the post mortem changes i n i so la ted collagen f i b e r s i n sect ion mater ia l but we have not looked a t the differences between post mortem muscle samples. techniques involved i n preparation a r e pa r t i cu la r ly important. We need t o break t h i s muscle t i s s u e apar t i n the proper place t o ge t the perspective of t h e connective t i s s u e s . A s it looks now i n sha t te r ing the muscle, you don't r e a l l y have much cont ro l of t h e s i t e of breakage. I th ink we need t o devise some technique f o r t ea r ing the muscle apar t t o look a t these connective t i s s u e s between ear ly and l a t e post mortem times, a f t e r enzyme treatments, and other var ia t ions .

I th ink t h a t the

M. H . Stromer, Iowa S ta t e University: 1 w o u l d l i k e t o respond t o D r . Kauffman's comment. animal and I have modified various surg ica l clamps tha t a r e avai lable on the market. One can maintain a relaxed sarcomere length without too much d i f f i c u l t y . The problem t h a t one ge ts i n t o i s t h i s : When you subject an i n t a c t piece of muscle with i t s exci table membranes and i ts load of AT" t o the temperature shock t h a t necessar i ly accompanies biopsy, you, of course, cause depolarization and a r e su l t an t shortening.

It i s possible t o do a biopsy on a l i v ing

M . D . Judge: Thank you. Let's move on t o the t h i r d paper. D r . Goll, may we have your summary comments?

D. E . G o l l : I have a s l i d e or two t o show an example of t he use of isopycnic centr i fugat ion f o r separation of subcel lular components. This i s a tube t h a t has had a microsomal f r ac t ion from muscle layered on top and centrifuged f o r 1 5 hours a t 25,000 FEN. f r ac t ion has been separated in to three bands: band labeled number 1, band labeled number 2, and then a contaminating band. bands were i so la ted and studied biochemically. The contaminating band contained a l l t h e mitochondria t h a t were o r ig ina l ly present as an impurity i n the microsomal f r ac t ion . graph of t he mater ia l i n band number 1. approximately 37,000 magnification. t h a t we have T tubules with the lateral c i s te rnae on e i the r s ide of them.

This microsomal

These th ree

The next s l i d e w i l l show a micro- These micrographs a r e a l l a t

You w i l l see areas where it appears

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These, i n f ac t , a l s o m i g h t be fragments of T tubules t h a t have been broken by homogenization; consequently that upper band labeled number 1 seem t 3 contain T tubules and a l so l a t e r a l c i s te rnae . It was of i n t e re s t t h a t th i s band did not bind any calcium, suggesting t h a t nei ther l a t e r a l c is ternae nor T tubules possess calcium accumulating a b i l i t y . preceding s l ide , a r e sarcolemma membranes t h a t have been fragmented by passing through a meat gr inder . The next s l i d e shows a n e lectron micrograph of tha t f r ac t ion labeled number 2. homogenous looking f i e l d . This i s negatively s ta ined a t t h e top and pos i t ive ly s ta ined a t the bottom. and i s apparently sarcoplasmic r e t i c u l a r membranes .

Also found i n t h i s f r ac t ion t h a t was labeled number 1 i n the

This is a much more

This mater ia l does accumulate calcium

R . M . Robson: I was wgndering what would be a r e l a t i v e proportion i n a muscle c e l l of t h e T tubule plus l a t e r a l c is ternae compared t o the SR?

D . E . Gol l : Other workers found three p a r t s by weight of t h e SR f rac t ion as compared t o one part by w e i g h t of t h e T tubules .

C . E . Allen: With the isopycnic centrifbgation, do you think there is a poss ib i l i t y of applying t h i s t o things l i k e f a t ce l l s ?

D. E. Goll: I would guess t h a t you would have t o s tar t w i t h layering the gradient on top of t h e sample. You would s t a r t almost i n reverse with the mater ia l a t the bottom and then centrifuging it up. You would probably separate d i f f e ren t populations of f a t c e l l s according t o t h e i r f a t content.

R . G . Cessens, University of Wisconsin: Would you comment on t he use of enzymes such a s collagenase a s a prestep t o the centr i fugat ion tha t you described?

D . E . Gol l : There has been one report t h a t I know of using collagenase t 3 "soften up" muscle c e l l s p r io r t o homogenization so you don't have t o subject t he c e l l t o a meat grinder o r something so severe t o d is rupt t he c e l l . m y s e l f because I would be concerned about t he presence of contaminating proteases and t h e i r e f fec t on the c e l l membrane. It i s d i f f i c u l t t o purify collagenases although I understand that purif ied sepa ra t ims a r e now ava i lab le .

Apparently it worked. I have never used collagenases

R . A . Merkle, Michigan S ta t e University: I have a question f o r D r . Robson. Based on t h e e f f ec t that the CAF f ac to r had on tropomyosin, a r e you thinking along t h e l i n e s that the Z l i n e d is in tegra t ion m i g h t be an e f f e c t on t h e tropomyosin ra ther than alpha act inin?

R . M . Robson: We a re thinking along severa l l i n e s as a matter of f a c t . Since t h e cmple te u l t ra -s t ruc ture of the Z d i sc i s not known f o r cer ta in , and s ince we don't know how f a r tropomyosin and troponin go i n t o t h e s t ruc ture i t s e l f , it i s qui te possible that ne i ther ac t in

nor alpha ac t in in are a l t e r ed but ra ther s l i g h t poss ib i l i t y which we have not ye t i s t h a t there i s a very small amount of i s not widely recognized as nryofibril lar

a r e re leased. There i s another completely ruled out and t h a t another protein present which protein .

R . A . Merkel: I f you accept t he idea of an e f f ec t on tropomyosin, then how do you r e c t i f y the differences between red and white f i b e r types, s ince there i s much l e s s d i s in tegra t ion of t he Z l i n e i n red f i b e r s during aging as commred t o white f ibers?

R. M . Robson: I would explain t h a t primarily not from a difference i n the Z l i n e i t s e l f but from a difference i n the amount of the calcium act ivated f ac to r present i n the d i f f e ren t types of c e l l s . That might be the most l og ica l explanation.

Id. W . Migura, Wilson and Company: If enzymic a c t i v i t y can be characterized within one pa r t i cu la r subunit of t he protein and t h a t subunit subsequently eluted, i s the enzymic a c t i v i t y negated by the use of SDS?

R . M. Robson: Yes, of course, it would be. When y3u add SDS you denature the pro te in so you would lose the a c t i v i t y . of ac tua l ly preparing the protein and s t i l l having it ac t ive you would have t o electrophorese it i n the absence of SDS and then work w i t h t h a t f r ac t ion .

If you a r e thinking

M . L. Greaser: There is ac tua l ly a method of removing SDS from proteins and it has been shown that cer ta in enzymes can, i n f a c t , be renatured a f t e r being put i n SDS. It involves passing the purif ied protein i n a mixture w i t h SDS through an ion exchange r e s in a n 4 i n many cases, one can recover 75 t o 9 6 of t h e enzymatic a c t i v i t y , so SDS i s not necessar i ly a permanent denaturing agent but i n t he presence of SDS, of course, it is unfolded.

R. M . Robson: I d idn ' t mean t o imply t h a t it was t o t a l l y impossible. The reference he i s t a lk ing about i s Weber and Kooder i n JBC, 1971.

C . E . Allen: I ' d l i k e t o ask D r . Dutson if, i n the scanning scope, you can dis t inguish between collagen, r e t i c u l i n , and e l a s t i n f ibe r s?

T . R . Dutson: No, I r e a l l y don't th ink there i s any way unless you a re able t o l a b e l one protein with something l i k e s i l v e r s ta in ing or some electron dense substance and then take a scanning electron micrograph o r secondary electron image and use the electron microprobe t o pick up that spec i f i ca l ly s ta ined substance. I n t h i s way you might be able t o ge t t he s t r u c t u r a l re la t ionship of those two. I n OUT work thus f a r , a l l we can see is f ibrous networks which m i g h t not even be collagen, e l a s t i n or r e t i c u l i n . They might just be other proteins or mucopolysaccharide-protein complexes t h a t are i n t h e ground substance connecting t h e other f i b r i l l a r prokeins. All of the proteins i n this fjxed s t a t e would appear r a the r f ibrous so it i s d i f f i c u l t t o say a t t h i s point .

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B. B. Marsh: I n opening t h i s session on "Muscle Membranes and Meat Properties," I would f i rs t l i k e t o express my thanks t o t h e committee who have sa ably ass i s ted me. Their names a r e l i s ted i n the back of your program, and I would pa r t i cu la r ly mention the cooperation I received from Fred Parr ish, the coordinator of t h i s sessim.

To t h e i n i t i a t e d , membranes a r e a n exci t ing area; t o the uni t ia ted , they a re a very complex and involved f i e l d f u l l of sometimes incomprehen- s i b l e j a r g m . For t h i s reason we feel it i s pa r t i cu la r ly appropriate t h a t t he top ic should be discussed a t length today. going t o control meat qual i ty , it i s c lear t h a t we must f i r s t understand meat and, of course, t h e muscle from which it i s derived. The empirical try-it-and-see methods of improving meat qua l i ty have j u s t about had t h e i r day. From now on we've got t he d i f f i c u l t problems t o solve--the ones which are not amenable t o t h e d i r e c t t r ia l -and-error approach and w e hope t h a t our topic today w i l l l a y the groundwork f o r a phase of renewed vigor i n t h i s a rea .

If we a r e ever

We've assembled four outstanding workers today, and I ' m personally very g ra t e fu l t o them f o r agreeing t o present papers under t h i s general t i t l e . i s t o be given by Dr . Marv Stromer of Iowa S ta t e University.

"he f i rs t paper, "The Structure and Function of C e l l Membranes,"