682 R. F. GLASCOCK AND W. G. HOEKSTRA I959one of us (W. G. H.) was on leave from the Departmentof Biochemistry, University of Wisconsin, Madison, Wis-consin, U.S.A. as a Merck Senior Postdoctoral Fellow ofthe National Academy of Sciences, National ResearchCouncil, Washington D.C.

REFERENCES

Asboe-Hansen, G. (1958). Physiol. Rev. 38, 446.Budy, A. M. (1955). Arch. int. Pharmacodyn. 103, 435.Cantarow, A., Rakoff, A. E., Paschkis, K. E., Hansen, L. P.& Walkling, A. A. (1943). Proc. Soc. exp. Biol., N. Y., 52,256.

Courtice, F. C. (1943). J. Physiol. 102, 290.Dodds, E. C., Folley, S. J., Glascock, R. F. & Lawson, W.

(1958). Biochem. J. 68, 161.Dodds, E. C., Golberg, L., Grunfeld, E. I., Lawson, W.,

Saffer, C. M., jun. & Robinson, R. (1944). Proc. Roy.Soc. B, 132, 83.

Dodgson, K. S., Garton, G. A., Stubbs, A. L. & Williams,R. T. (1948). Biochem. J. 42, 357.

Emmens, C. W. (1940-41). J. End,ocrin. 2, 448.Folley, S. J. (1956). The Physiology and Biochemistry of

Lactation. Edinburgh and London: Oliver and Boyd.Glascock, R. F. (1954a). Radioisotope Conference, vol. 1,

p. 262. Ed. Johnston, J. E., Faires, R. A. & Millett,R. J. London: Butterworths Scientific Publications.

Glascock, R. F. (1954b). Isotopic Gas Analysis for Bio-chemists. New York: Academic Press Inc.

Glascock, R. F. (1956). Proc. Int. Conf. Peaceful Uses ofAtomic Energy, 12, 397. New York: United Nations.

Glascock, R. F. & Hoekstra, W. G. (1958). Proc. 2nd Int.Conf. Peaceful Uses of Atomic Energy, (in the Press).New York: United Nations.

Green, S. (1954). Proc. Soc. exp. Biol., N. Y., 86, 653.Hanahan, D. J., Daskalakis, E. G., Edwards, T. & Dauben,H. J., jun. (1953). Endocrinology, 53, 163.

Joseph, N. R., Engel, M. B. & Catchpole, H. R. (1954).Arch. Path. 58, 40.

Kalman, S. M. (1955). J. Pharmacol. 115, 442.McCorquodale, D. J. & Mueller, G. C. (1958). J. biol. Chem.

232, 31.Rapp, G. W. & Richardson, G. C. (1952). Science, 115, 265.Robinson, T. J., Moore, N. W. & Binet, F. E. (1956).

J. Endocrin. 14, 1.Sandberg, A. A. & Slaunwhite, W. R., jun. (1957). J. clin.

Invest. 36, 1266.Smith, A. E. W. & Williams, P. C. (1948). Biochem. J. 42,

253.Szego, C. M. (1957). In Physiological Triggers, p. 152. Ed.by Bullock, T. H. Washington, D.C.: American Physio-logical Society.

Turner, C. W. (1956). J. Anim. Sci. 15, 13.Twombly, G. H. (1951). Acta Un. int. Cancr. 7, 882.Twombly, G. H. & Schoenewaldt, E. F. (1951). Cancer, 4,

296.Wiberg, G. S. & Stephenson, N. R. (1957). Canad. J.

Biochem. Physiol. 35, 1107.

A Ribonucleoprotein of Skeletal Muscle and its Relation to the Myofibril

BY S. V. PERRY AND M. ZYDOWO*Department of BiocheMi8try, Univer8ity of Cambridge

(Received 12 February 1959)

From the work of a number of investigators thereis evidence for the association of nucleic acid withpreparations of certain myofibrillar proteins. Thisproperty is particularly marked in tropomyosin,which can be obtained from a variety of species andmuscle types as a crystallizable complex, nucleo-tropomyosin, containing up to 20 % of nucleic acid(Hamoir, 1951 a, b; Sheng & Tsao, 1954). Smalleramounts of ribonucleic acid have been reported tobe present in purified myosin preparations(Mihalyi, Laki & Knoller, 1957; Mihalyi, Brodley &Knoller, 1957). The significance of the ribonucleicacid associated with these proteins has been amatter for speculation, but the recent finding(Perry & Zydowo, 1958, 1959) that a ribonucleo-protein is one of the components of the extraprotein (Szent-Gyorgyi, Mazia & Szent-Gy6rgyi,1955) suggests that the myofibrillar fraction of the

cell may be the origin of the ribonucleic acid. Thispaper is concerned with the characterization of theribonucleoprotein extracted from the myofibriland its relation to the distribution of ribonucleicacid in the muscle cell.

METHODS

Preparation of myofibrils and mnucle granule8. Myo-fibrils were prepared from the back and leg muscles of therabbit and the breast muscles of the hen by the method ofPerry & Grey (1956), modified as described by Perry &Zydowo (1959). Washed granular preparations were pre-pared from the same sources by the method of Perry &Zydowo (1959).

Preparation of ribonucleoprotein. The dilute extra-proteinfraction, prepared as described previously (Perry &Zydowo, 1959), in 0-04M-KCI containing 6-7 mM-potassiumphosphate buffer, pH 7-2, was adjusted to 0 1M-KCI by theaddition of solid KCI and to pH 7-6 with M-2-amino-2-hydroxymethylpropane-1:3-diol (tris). This solution wasrun into a column of diethylaminoethylcellulose equili-brated with 01M-KCI containing 0-02M-tris chloride

* Research Fellow of the Rockefeller Foundation 1957-8.Present address: Department of Biochemistry, MedicalAcademy, Gdansk, Poland.

RIBONUCLEOPROTEIN OF MUSCLEbuffer, pH 7-6, and the ribonucleoprotein eluted between0-4 and 1-5M-KC1 was collected. This is fraction IV ofPerry & Zydowo (1959). The eluted ribonucleoprotein wasexhaustively dialysed against distilled water, freeze-driedand stored at - 150. All preparations and chromatographicseparations were carried out at 10 to 20.

Fractionation of muscle. A weighed sample of mincedfresh muscle was homogenized for 40-60 sec. in a smallhomogenizer of the Waring Blendor type with a knownamount of medium (usually about 10 vol. of 0-25M-sucroseor 01M-KCl-borate buffer, pH 7-1). From weighedportions of the homogenate, each containing about 2 g. ofmuscle, the following fractions were isolated.

(1) Nuclear-myofibrillar. This was the residue sediment-ing after 15 min. at 800g, from which soluble proteins(sarcoplasm) and granular material were removed by sus-pending the residue from the first centrifuging in freshmedium and washing by centrifuging three times. (2)Granular. This fraction was sedimented by centrifuging for2 hr. at 15 000 g the combined supernatant and washingsseparated from the nuclear-myofibrillar fraction. It waswashed once by centrifuging again. (3) Sarcoplasmic. Thisfraction consisted of the combined supernatant and wash-ings after removal of the granular fraction. It containedsmall amounts of granular material which could besedimented by prolonged centrifuging at higher speeds.Fractionations were usually carried through in triplicate.

Chromatography. Diethylaminoethylcellulose was em-ployed as previously described (Perry & Zydowo, 1959).The sample of ECTEOLA-cellulose used (0-25 m-equiv. ofN/g.) was prepared by Dr J. P. Waller in this Laboratoryby the method of Peterson & Sober (1956). Samples forfractionation on ECTEOLA-cellulose were equilibrated bydialysis against 0-01 M-potassium phosphate buffer, pH7 05, and applied to a column of the cellulose prepared andequilibrated against the same buffer, as described fordiethylaminoethylcellulose (Perry & Zydowo, 1959). Thecolumn was developed by applying a gradient of KCI dis-solved in 001 M-potassium phosphate buffer, pH 7.05.Chromatography was carried out at 1-2°. The chlorideconcentration of the eluate was estimated by titration with0-02N-AgNO3 (Perry & Zydowo, 1959). Application of KCIup to molar concentration eluted about 87% of the totalmaterialabsorbing at 260mSt that was applied. M-KOHwasrequired to elute the remaining material from the column.

Es8timation of bases in ribonucleoprotein. Separation ofbases was achieved by hydrolysis of the nucleoprotein in72% (w/w) perchloric acid, followed by chromatographyon Whatman no. 1 paper as described by Wyatt (1951)with the modification that the hydrolysis was carried outfor 2 hr. as recommended by Markham (1955). The amountsof the bases eluted from the paper were estimated spectro-photometrically.

Estimation of nucleic acids. Whole muscle, rapidly re-moved from the animal and chilled in ice, was prepared foranalysis by dissecting it free from obvious connective tissue.A portion (0-5-1.0 g.) of muscle was homogenized in theMSE Masticator (Measuring and Scientific Equipment Ltd.,London) with 20 vol. of absolute ethanol, the homogenatewas transferred quantitatively to a centrifuge tube andusually washed twice with ethanol and two or three timeswith ether. Whole homogenates, prepared at 00 with a smallhomogenizer of the Waring Blendor type, myofibrils andtissue fractions were precipitated with at least 4 vol. ofethanol and subsequently washed by centrifuging twice

with ethanol and twice with ether. In some instances thehomogenates of whole muscle and the fractions obtainedfrom them were precipitated at 00 with an equal volume of15% (w/v) trichloroacetic acid before washing withethanol. The air-dried residues were extracted three timesat 00 with about 50 vol. of 2% (w/v) perchloric acid toremove nucleotides. These extracts were discarded andnucleic acids subsequently extracted four or five times with30-50 vol. of 10% (w/v) perchloric acid at 700. The com-pleteness of the extraction of both nucleotides and nucleicacid was ensured by measurement of absorption of theextracts at both 260 and 280 m1t.

Total P analysis was carried out by digesting up to 2 ml.of the 10% perchloric acid extracts with 0-5 ml. of 1ON-H2SO4. When the contents of the tubes had been concen-trated to small volume by an initial heating, digestion wascontinued for 30 min. on an electric furnace to drive off theperchloric acid, and P estimated under the Fiske & Sub-barow (1925) conditions.

Ribose was estimated on the 10% perchloric acid ex-tracts by the orcinol method as modified by Ceriotti (1955).Ribose values were converted into ribonucleic acid P valueswith a factor determined by analysis of the 10% perchloricacid extract of a pooled sample of the ribonucleoproteinisolated from myofibril preparations (see Results section).The extinctions at 260 and 268 m,u of the 10% per-

chloric acid extracts were also measured but these valuesdid not agree well with the amounts of ribose and Ppresent. This was probably due to the extraction of someprotein by the 10% perchloric acid. Occasionally with theorcinol reaction a brown colour developed instead of theusual green characteristic for ribose. This was especiallynoted during analyses of whole dorsal muscle, and of sarco-plasmic and granular fractions from this muscle. Inter-ference was never observed with the myofibrillar fraction.It is possible that this interference was due to other carbo-hydrates, probably glycogen (Brown, 1946).The Dische (1930) reaction for deoxyribonucleic acids

was found to be too insensitive for most of the 10%perchloric acid extracts.

Other analytical methods. Tropomyosin was assayed bymeasuring the drop of viscosity under the influence of KCIas described by Perry (1953) and Perry & Corsi (1958),and adenosine triphosphatase activity was measured by thestandard procedure used in this Laboratory (Perry & Grey,1956).Total N was estimated by the usual micro-Kjeldahl

procedure with selenium oxide-CuSO4 mixture as catalyst.Absorption spectra in the ultraviolet range were obtained

with the Beckman Ratio Recording Spectrophotometer.Material&. All the chemicals used for analytical purposes

were of A.R. grade. Orcinol was recrystallized several timesfrom benzene after shaking with charcoal. isoAmylalcohol was redistilled before use. Tris was recrystallizedtwice from ethanol. Yeast sodium ribonucleate (BritishDrug Houses Ltd.) was purified according to the method ofWoodward (1944).

RESULTS

Composition of ribonucleoprotein fractionAs isolated from the extra-protein fraction ofrabbit myofibrils by chromatography on diethyl-aminoethylcellulose, the ribonucleoprotein (frac-

VoI. 72 683

S. V. PERRY AND M. ZYDOWO

tion IV, Perry & Zydowo, 1959) was soluble inwater and when freeze-dried from salt-free solu-tions it was readily redissolved at pH 7 0.Owing to difficulties in isolating sufficient

quantities it was not possible, however, to establishprecisely by rigorous tests the degree of homo-geneity of the ribonucleoprotein fraction. Bygradient-chromatography of the extra protein on

diethylaminoethylcellulose the nucleoprotein was

consistently eluted, at pH 7 6 in 0 02M-tris chloridebuffer, as a sharp, fairly symmetrical peak at a

chloride concentration ofabout 0 7 equiv./l. (Fig. 1).Analysis of the fraction as eluted from diethyl-aminoethylcellulose between 0-4 and 1-5M-KCI, thestandard procedure used for preparation of theribonucleoprotein, gave N/P ratios ranging from3-8 to 4-4. In the ultracentrifuge a sample obtainedby combining the ribonucleoprotein isolated fromthree different preparations of extra protein gave

a relatively slowly moving peak (S20, w. 2-6), fromwhich a small faster component had partly emergedafter 2 hr. at 59 000 rev./min. in 0 1M-KCI-0-05M-potassium phosphate buffer, pH 7-1.The presence of protein in fraction IV was

evident from the biuret test and the fact that theminimum of the ultraviolet-absorption curve

(Fig. 2) was in the region of 240 m,u rather than at230 m,, the minimum characteristic of pure ribo-nucleic acid (RNA) (Beaven, Holiday & Johnson,

14

1-2

E

'14

1955). There was some suggestion of partial frac-tionation of the ribonucleoprotein by diethyl-aminoethylcellulose for the wavelength at the pointof minimum absorption in the ultraviolet lightdecreased in samples taken in sequence as theribonucleoprotein was eluted from the column byprogressively increasing the KC1 concentrationfrom 0 4 to 1-5M. Minimum values approaching230 m, were reached at the peak of the elutionchromatogram, but samples at all stages of theelution contained nucleic acid, for in every case

the ratio E260/E280 was appreciably greater thanone.

Much better separation of protein and RNA was

obtained when the ribonucleoprotein was rechro-matographed on ECTEOLA-cellulose (Peterson &Sober, 1956). Fraction IV dissolved in 10 mm-potassium phosphate buffer, pH 7-05, was held on

ECTEOLA-cellulose and subsequent gradient-elution with KCI gave two peaks (Fig. 3). The firstand smaller peak, as judged by absorption at 280and 260 mj., was eluted at a chloride concentrationof 0.1 equiv./l., and the second at 0 3 equiv./I. Forthe first peak the ratio E26o/E280 barely exceeded 1,which from the data obtained by Warburg &Christian (1941-42) with enolase would indicatethat less than about 5 % ofnucleic acid was present.

08

1-4

1 2

.,

-rv

0-8 vL.

00,6 'C

04 oU

Vol. of eluate (ml.)

Fig. 1. Gradient-elution of the nucleoprotein from rabbitmyofibrils on diethylaminoethylcellulose. Extra protein(72-5 ml.; Elcm. 13-2 at 280 m,) was concentrated byprecipitation with saturated (NH4),SO4, and was dis-solved in 0-1M-KCl-0-02M-tris chloride buffer, pH 7-6,and applied to a column 27 cm. x 2 cm. diam. The bulk ofthe protein was previously removed by elution with0 4M-KCl-0 02m-tris buffer, and a gradient to 2-Om-KCl-002M-tris buffer was applied at the point A.0, E280 ml,; chloride concentration.

E

. 0-4Li4

Wavelength (mp)

Fig. 2. Ultraviolet-absorption spectrum of the nucleo-protein isolated from rabbit myofibrils; in 1-5M-KCI-0-02M-tris chloride buffer, pH 7-6.

684 I959

RIBONUCLEOPROTEIN OF MUSCLE

The peak eluted at higher salt concentration hada ratio E260/E280 about 2 and was relatively pureRNA, although the minimum of the absorptioncurve was not exactly at 230 mp, which suggestedthat small amounts of protein were still present.

Ribonucleic acid componentEarlier investigations (Perry & Zydowo, 1959)

indicated that there was no significant amount ofdeoxyribonucleic acid in the ribonucleoproteinfraction. Quantitative chromatographic separationof the bases liberated by hydrolysis in 72 % (w/w)perchloric acid (Wyatt, 1951) showed the composi-tion of ribonucleic acid obtained from myofibrilsisolated from the mixed back and leg muscles ofthe rabbit to be relatively constant. These resultsare summarized in Table 1, in which is also in-cluded the base analysis of the correspondingribonucleoprotein isolated from myofibrils pre-pared from hen-breast muscle. The composition ofthe RNA is remarkably similar in both species andis characterized by a high-guanine and low-uracilcontent, features which are characteristic of RNAfrom most animal species.

Yeast nucleic acid which has been partly de-graded by ribonuclease is, however, also character-ized by a relatively high guanine content (Maga-sanik & Chargaff, 1951; Markham & Smith, 1952;Volkin & Cohn, 1953), and it was suggested byDr R. Markham that the high content of thispurine in the myofibrillar RNA might be indicative

of partial degradation of the muscle nucleic acid byaction of intracellular enzymes during preparation.It seemed very unlikely that much degradation ofthe myofibrillar RNA had occurred, for all pre-parations were made at 00 and the ribonucleaseactivity of skeletal muscle is known to be very low(Bain & Rusch, 1944; Greenstein & Thompson,1943; Roth & Milstein, 1952). As a control experi-ment a solution of yeast RNA, previously purifiedto remove degraded material, was incubated for2 hr. at 250 with a rabbit skeletal-muscle homo-genate made in 041 M-KCI containing 39 m;-borate buffer, pH 7-1 (Perry, 1953). No significantincrease in acid-soluble material absorbing at260 m,u could be detected after this incubation, andthe ratio of the bases present in the acid-insolubleyeast RNA was unchanged.

Excellent agreement was obtained between theamount of ribose estimated by the orcinol methodper ,ug.atom of total phosphorus in the ribonucleo-protein and that predicted from the base analysis.On the basis that only ribose which is purine-linkedis estimated under the conditions of the orcinolreaction, the analyses in Table 1 indicate that forevery pg.atom of rabbit RNA phosphorus, 0-532 ,u-mole of ribose should be estimated by the orcinolreaction. With the sample used as the standard inthese studies 0-531 ,umole of ribose was in factestimated/,ug.atom of total RNA phosphorus.Analysis of another sample of the ribonucleoproteingave a value of 0-527 ,umole of ribose/,ug.atom oftotal RNA phosphorus.

E

WU

06 _

- 05 5CT

O._.03 o02

01 '0oiU~

Fig. 3. Chromatography of ribonucleoprotein onECTEOLA-cellulose. A sample (2 ml.) of ribonucleo-protein solution (E1 cm, 17-62 at 260 m,u) in 0-01 M-potasium phosphate buffer, pH 7 05, was applied to 1 g.of the cellulose in a column (1 cm. diam.). Gradient to0-7M-KCl-OOlM-phosphate was applied at the point A.O, E280 mu; .- -, E260 mp,; *, chloride concentration.

Protein componentWith the values for total nitrogen (16.74) and

phosphorus (9.57) contents of the myofibrillarRNA calculated from the base analyses (Table 1),figures can be obtained for the non-RNA nitrogenin the ribonucleprotein from the data on elemen-tary analyses quoted earlier. Assuming that all thephosphorus present is derived from RNA, thenucleic acid accounts for 41-47 % of the totalnitrogen. The additional nitrogen was presumed tobe due to protein, as it was non-dialysable, the

Table 1. Ba8e compo8ition of ribonucleoproteini8olated from the extra-protein fraction of skeletalmyoftbril8Results are expressed in moles/100 moles of total bases.

Results for the rabbit are averaged from seven estimations,made on three different samples; those for the hen are fromfour estimations on the same sample. Results are given+ S.E.M.

GuanineAdenineCytosineUracil

Rabbit35-6±0-5917-6+0-3428-6±0-5518*2±0-61

Hen35-1 ±07519-9±0-2128-8±0-7816-2±0-10

Vol. 72 685

S. V. PERRY AND M. ZYDOWO

material was biuret-positive and possessed thegeneral properties of protein. In one experiment32 % of the total N was not extracted by hot 10 %perchloric acid, suggesting that at least this pro-portion of the nitrogen was due to protein. Thefractionation on ECTEOLA-cellulose indicated aprotein/RNA ratio comparable with that obtainedfrom the nitrogen and phosphorus analyses, in sofar as it was estimated that 50 % of the totalmaterial eluted was in the peak eluted at the lowerionic strength.The nature of the protein component was not

established but the chromatographic behaviour ofthe bulk of the sarcoplasmic components, myosin,actin and tropomyosin (Perry & Zydowo, 1959;Perry, 1959, unpublished work), all of which wereeluted from diethylaminoethylcellulose at pH 7 6 byKCI at concentrations below 04M, suggested thatit was not identical with any of these. In view ofthe fact that both tropomyosin and myosin pre-parations have been reported to contain RNA thepossibility of the identity of the protein moietywith either of these myofibrillar proteins wasfurther investigated. After equilibration bydialysis against 78 mM-borate buffer, pH 7-1(Perry & Corsi, 1958), the solution of a pooledsample of ribonucleoprotein was slightly viscous,relative viscosity at 00 = 1L1 (E1lcm 25-33 at260 mv). A sample of yeast RNA of similar ex-tinction, which had been purified to remove depoly-merized material by the method of Woodward(1944), had a relative viscosity of 1 01. Addition ofKCI to 0-5M reduced the relative viscosity of theribonucleoprotein to 1-05, whereas the viscosity ofyeast nucleic acid was virtually unchanged. A fallin viscosity on increasing the ionic strength ischaracteristic of tropomyosin. Nevertheless, theviscosity effects obtained with the ribonucleopro-tein were very small and certainly not largeenough to be used for reliable assays of tropomyo-sin, but if the whole fall in viscosity was due to thisprotein the amount present did not account formore than 10-15 % of the non-nucleic acid nitrogen.It can be concluded that tropomyosin, if indeedpresent at all in the ribonucleoprotein fraction, isonly a minor component.The absence of any adenosine triphosphatase

activity and the solubility characteristics of theribonucleoprotein strongly suggested, in addition

to the facts given above, that myosin was not theprotein associated with the nucleic acid.

Origin of the ribonucleoproteinThe ribonucleoprotein was found to be present

in the extra-protein fraction of myofibrils isolatedfrom rabbit skeletal muscle and from breast muscleof the hen, the only other species so far studied.This fact, coupled with the results of RNA deter-minations on isolated myofibrils (see Table 2),indicated that the ribonucleoprotein was a con-sistent and significant component of all myofibrilpreparations.On extraction of myofibrils with the pyrophos-

phate-KCl solution (Hasselbach & Schneider, 1951)used in the preparation of the extra protein, about60 % of the RNA, and protein consisting mainly ofmyosin and representing in all about 70 % of thatpresent in the myofibril, passed into solution. Asmost of the RNA, however, stayed in solutionwhen the (acto)myosin was subsequently pre-cipitated from this extract it seemed that underthese conditions at least there was not a particu-larly strong affinity between the two proteins. Onthe other hand, the bulk of the actin and tropo-myosin could be extracted from myofibrils bytreatment with 5 mM-tris chloride buffer, pH 8-1(Perry & Corsi, 1958), accompanied by only about1.5 % of the myofibrillar RNA.Formed elements in the cell which are known to

contain ribonucleoprotein and which would con-ceivably contaminate the myofibrillar fraction arenuclei and the endoplasmic reticulum. Althoughnuclei are seldom seen in myofibril preparations thelatter contained 630-970 /tg. of deoxyribonucleicacid phosphorus/g. of total nitrogen, indicatingthat appreciable contamination was present. Theratio of RNA to deoxyribonucleic acid (DNA) innuclei does not, however, exceed 1: 4 (Dounce,1955), and it can be concluded from the RNA anddeoxyribonucleic acid concentrations in myofibrilsthat probably not more than about 10% of thetotal RNA is of nuclear origin.

Rabbit skeletal muscle contains few granulescomparable in size with the mitochondria of liverand of the more oxidative muscles. Under condi-tions adequate to sediment the mitochondria inthese tissues extremely low yields of granules areobtained from rabbit-muscle homogenates, and it is

Table 2. Ribonucleic acid and total nucleic acid content of whole muscle and myoflbril8 from the rabbit

S.E.M. values are given when analysis has been carried out on more than two different animals or preparations. Figuresin parentheses indicate the number of determinations.

Whole psoas (,ug./g. wet wt.)Whole latissimus dorsi (ILg./g. wet wt.)Myofibril preparation (,ug./mg. of total N)

Ribose262+27-8 (4)210 (2)4 37+0 59 (3)

RNA phosphorus102+10 8 (4)81 (2)1*70+0-23 (3)

Total nucleicacid phosphorus123+10-9 (4)113±14-4 (5)2-81 (2)

686 I959

RIBONUCLEOPROTEIN OF MUSCLEdifficult to decide upon the appropriate conditionsto sediment the mitochondrial fraction to separateit from the fraction corresponding to the microsomefraction of other tissues.Although few granules were apparent in washed

myofibril preparations, the possibility was con-sidered that the myofibrillar ribonucleoproteincould arise from contamination with the smallgranules which can be seen on microscopic exami-nation of sarcoplasmic extracts. The granulesprepared from a muscle homogenate in 0- 1 M-KCI-39 mM-borate buffer, pH 7-1, by centrifugingfor 2 hr. at 15 000 g were extracted in the sameway as for the preparation of the extra protein, andthe resulting solution was chromatographed ondiethylaminoethylcellulose in the manner used tofractionate the extra protein (Perry & Zydowo,1959). Fig. 4 illustrates the result ofsuch an experi-ment and shows the relatively small amount ofribonucleoprotein which was extracted from thesegranules compared with that from myofibrilstreated in the same way (Perry & Zydowo, 1959).It was concluded that even if all of the granulessedimenting after 2 hr. at 15 000 g, which wereoriginally present in the muscle, remained with themyofibrils during their preparation, the granuleswould account for only about one-fifth or one-sixthof the total ribonucleic acid usually found in theextra-protein fraction. This clearly excluded con-tamination with the larger granules as a source ofthe myofibrillar ribonuclear protein, as do also thefractionation studies described below.

Information about the endoplasmic reticulumhas been obtained mainly with liver cells and

1-2

1-0

08

w 0-6L&J

0-4

chemical data about its presumed equivalent inmuscle cells, the sarcoplasmic reticulum, is lacking.In most cells the bulk of the cytoplasmic ribo-nucleoprotein is associated with the endoplasmicreticulum fraction, which requires prolonged centri-fuging at high speeds for sedimentation (Loftfield,1957). High-speed centrifuging of sarcoplasmicextracts from rabbit skeletal muscle did not yielda microsome fraction containing the bulk of theRNA of the cell (see below), but most of the RNAsedimented at low speeds with the myofibrillar-nuclear fraction. The myofibrillar ribonucleopro-tein did not arise from contamination with thesarcoplasmic proteins which are still present in well-washed myofibrils, for little RNA could be demon-strated in the sarcoplasmic fraction either byanalysis or chromatography. When the fraction ofthe cytoplasm not sedimented after 2 hr. at15 000 g (consisting of the soluble proteins andprobably some sarcoplasmic reticulum) is fraction-ated on diethylaminoethylcellulose no significantamounts of ribonucleoprotein are eluted at higherKCI concentrations (Perry & Zydowo, 1959).

Distribution of nucleic acid within the muscle cellAlthough the values in the literature for the

nucleic acid content of muscle vary considerablyfrom author to author, in all cases the amountspresent are relatively low compared with thosepresent in other tissues. An earlier study (Perry,1952) indicated that significant amounts of RNAphosphorus were present in skeletal myofibril pre-parations, and Needham & Cawkwell (1957) havefound RNA in the fraction of rat uterus that is

12

1 0-

430-8 v

0-6 'o_

-C

v

00-4 c.

u

o0n

0 200 t400 600 800 t 1000A Vol. of eluate (ml.) B

0

Fig. 4. Chromatography on diethylaminoethylcellulose of extract of granules sedimented from a rabbit sarcoplasmicextract by centrifuging for 2 hr. at 15 000 g. The extract was prepared in the same way as the extra-protein fractionfrom myofibrils (see Methods). A sample (50 ml.) of protein solution (El Cm. 2-85 at 280 m,u) dissolved in 0-1 M-KCl-0-02M-tris chloride buffer, pH 7-6, was applied to a column (17 cm. x 2 cm. diam.). Gradients to 0-45M-KCI-0-02M-tris and to 1-5M-KCI-0-02M-tris buffer were applied at points A and B respectively. O, E280 mp; -- -, E260 mngi;*, chloride concentration.

VoI. 72 687

S. V. PERRY AND M. ZYDOWOinsoluble at lower ionic strengths, which presum-ably contains the contractile proteins of this tissue.More detailed investigation of the ribonucleic acidcontent of whole muscle and myofibril preparationindicated that even after repeated washings andcentrifugings the myofibrils contained ribonucleicacid, which, if it was all associated with thesestructures in situ, represented almost half thatpresent in the whole muscle (Table 2). SatisfactoryRNA ribose estimations were obtained with rabbitpsoas muscle, but estimations on whole longissimusdorsi muscle and the granular and sarcoplasmicfractions isolated from it frequently suffered frominterference owing to a brown which developedunder the condition of the orcinol reaction forribose. In consequence only very few satisfactoryvalues for the RNA ribose content of longissimusdorsi muscle were obtained. Nevertheless thesewere comparable with the values obtained for thepsoas. Considerable variation in values was notedbetween animals, as has also been reported for therat by Schmidt & Schlieff (1956).To discover more precisely how the nucleic acid

was distributed within the muscle cell, longissimusdorsi muscle, which formed the bulk of the tissueused for myofibril preparations, was homogenized

both in 0 25M-sucrose and in 0I1M-KCI-39 mM-borate buffer, pH 7d1, and separated into myofib-rillar-nuclear and so-called granular and sarco-plasmic fractions (see Methods). Table 3 indicatesthe distribution of total nitrogen between thesefractions and indicates the relatively small amountof nitrogen sedimenting after 2 hr. at 15 000 g,conditions far in excess of those required to sedi-ment mitochondria in other tissues. It is difficultto generalize about muscle from experience withother tissues, but some microsomes might beexpected to sediment in this granular fraction. Inone experiment, on further sedimentation of thesarcoplasmic fraction obtained by homogenizationin 0-1M-KCI-39 mM borate buffer, pH 7-1, anamount of material representing 1-7 mg. of nitro-gen/g. wet wt. of muscle was obtained after 1 hr. at100 000 g, conditions which would be expected tosediment the microsome fraction. It is worthnoting that the sarcoplasmic fraction was a smallerproportion of the total nitrogen when fractionationwas done in 0 25M-sucrose rather than in the ionicmedium.The total phosphorus determinations made on

hot 10% perchloric acid extracts of the fractionsclearly indicate that about 70% of the total nucleic

Table 3. Distribution of total nitrogen between fractions of homogenates of rabbit longissimus dorsi muscle

s.E.M. values are given when analysis has been carried out on more than two samples from different animals. Figures inparentheses indicate the number of determinations.

Homogenized in 0 I M-KCl-39 mM-borate bufferTotal nitrogen (mg./g. wet wt.)Percentage of total nitrogen

Homogenized in 0-25M-sucroseTotal nitrogen (mg./g. wet wt.)Percentage of total nitrogen

Myofibrils,nucleus Granules Sarcoplasm

20-6±0-42 (5) 1-7±0-15 (4) 15*2+0-14 (4)55 5 40

23.9 (2)66

1.3 (1)4

110 (1)30

Table 4. Di8tribution of nucleic acid between fractions obtained fromhomogenates of rabbit longissimus dorsi muscle

Results are expressed in pg./g. wet wt. of muscle and, except for the figures in parentheses, are average values obtainedfrom fractionations each carried out in triplicate.

Homogenized in 0-25M-sucrose

Expt. I RNA phosphorusi Total P of 10% perchloric acid extract

E RNA phosphorusE Total P of 10% perchloric acid extractTotal P, distribution (%)

Homogenized in 041 m-KCl-39 mm-borate buffer

Expt 3 IRNA phosphorusiTotal P of 10% perchloric acid extract

Expt 4 IRNA phosphorusx Total P of 10% perchloric acid extract

Total P, distribution (%)

(1) (2) (3)Myofibrilsnucleus Granules Sarcoplasm

65-368-157.782-574

49-059.755-663*168

(4) (5)Whole Recoverymuscle (1), (2) & (3)

4-29.5 9.7 87-36-1 - 82-6 -15*5 19-4 123-8 117-412 14

(4.9)9.9

23*718

13-811*0(16.5)14-514

81-086-8

96*1

77-780*6

101-3

688 I959

RIBONUCLEOPROTEIN OF MUSCLEacid was carried down with the myofibrillarfraction irrespective of whether sucrose or anionic medium was used for fractionation (Table 4).From the limited ribose determinations obtainedit appears that about 60% of the ribonucleic acid islikewise associated with the myofibrillar-nuclearfraction. These results apply in both ionic or non-ionic media; in fact rather more nucleic acid issedimented at low speeds when the homogenate ismade in 0-25M-sucrose than when it is made in theKCl-borate buffer medium.

DISCUSSION

In the rabbit skeletal-muscle cell a large part of thetotal RNA is associated with the myofibrillar-nuclear fraction obtained on low-speed centrifugingof homogenates of this tissue. As most of this RNAis still present in myofibrils which have been sub.jected to further purification and repeated washingby centrifuging, it appears that there is a fairlyintimate association between the nucleic acid andthese structures. The bulk of the myofibrillar RNAexists as a ribonucleoprotein which is present in theextra-protein fraction, and it seems likely that thisribonucleoprotein is the source of the nucleic acidwhich is present in preparations of myosin andnucleotropomyosin. The base composition of theribonucleoprotein isolated from the extra proteinis very similar to that of the nucleic acid bound tomyosin, which has been studied by Mihalyi,Brodley & Knoller (1957). It is difficult to comparemany of the properties of the two preparations, asin the latter work the ribonucleoprotein fractiondisplayed considerable heterogeneity, due nodoubt to the fact that it was prepared by some-what drastic techniques designed to denature pre-ferentially the myosin. Mihalyi, Brodley & Knoller(1957) suggest that a heat-stable protein present intheir preparation bears some resemblance totropomyosin.The base analyses of the ribonucleic acid com-

ponent from nucleotropomyosin of various fishmuscles (Hamoir, 1951 b; Tarr, 1958) are alsosimilar to those of the rabbit and hen myofibrillarnucleoprotein. It is likely that nucleotropomyosinis a complex oftropomyosin with ribonucleoproteinrather than with ribonucleic acid alone.From the limited studies carried out, the protein

moiety of the isolated nucleoprotein does notappear to be identical with myosin or tropomyosin,and in view of the ease with which the ribonucleo-protein can be separated by chromatography frommyosin (S. V. Perry, unpublished work) and tropo-myosin (Perry & Zydowo, 1959), its associationwith them need not indicate any special significanceother than that under conditions for their extrac-tion, the ribonucleoprotein is also extracted and

44

shows some non-specific affinity for both theseproteins.

It is difficult to determine the precise localizationof the ribonucleoprotein in the muscle cytoplasm.The results exclude nuclear contamination fromcontributing more than a small fraction of the totalmyofibrillar RNA, but do not allow us to decidewhether the RNA is built into the filamentousstructure of the myofibril or is preferentiallylocalized in some other cytoplasmic element suchas the sarcoplasmic retioulum which may beattached to the myofibrils or sedimented with them.A number of workers have produced cytoehemiealevidence for the localization of nucleic acid in themyofibrils. Clavert, Mandel, Mandel & Jacob(1949) claimed to show a specific histoohemical-staining reaction for RNA in the A band of myo-fibrils, whereas Caspersson & Thorell (1942)observed in the I band a concentration of materialabsorbing at 259 m, which could be due either tonucleotides or to nucleic acid. Further, Gerendas &Matoltsy (1948) isolated from muscle nucleoprotein-like material which they considered played apart in producing the characteristic birefringentpattern of the myofibril. None of these studiesdistinguishes between ribonucleoprotein materialactually built into the myofibril structure ratherthan material in other fine structures which maybe overlying it.The studies carried out on the RNA distribution

indicate that in muscle homogenates in 0-1 M-potassium chloride-39 mM-borate buffer, pH 7-1,or in 0-25mr-sucrose, the bulk of the cytoplasmicRNA does not sediment in a manner comparablewith that of the microsome fraction of liver. Onfractionation of muscle the myofibrillar-nuclearfraction contained about 60% of the RNA of thewhole muscle. Some of this nucleic acid may havebeen lost from myofibrils during the repeatedwashing and centrifuging employed to purify pre-parations, for the average RNA content of suchpreparations is somewhat lower than that of themyofibrillar-nuclear fraction. If this lower figure istaken, and assuming that 1 g. of fresh musclecontains 20mg. of myofibrillar nitrogen, about40% of the total RNA in the cell would be associ-ated with the myofibrils.

Electron-microscope studies have shown reti-cular material to be abundant and associated withthe myofibril in a characteristic fashion in certaintypes of muscle (Porter & Palade, 1957). This is amarked feature of Ambly8tomna skeletal muscle, butin rabbit skeletal muscle sarcoplasmic reticulum isrelatively scarce, judging from the electron-microscope studies in the literature, and thestructures which are characteristic of Ambly4etomrareticulum have not been reported. If there is anassociation of ribonucleoprotein-rich reticulum

Bioch. 1959, 72

689VoI. 72

690 S. V. PERRY AND M. ZYDOWO I959with the myofibril it would be expected to be sedi-mented at low speeds with the myofibrillar-nuclearfraction. Thus in the ease with which ribonucleo-protein-rich material can be sedimented fromhomogenates muscle resembles hen oviduct tissue(Hendler, 1956) but differs from most animaltissues.The relatively low ribonucleic acid content of

muscle agrees with the observation (K. R. Porter,personal communication) that small granules com-parable with the ribonucleoprotein-rich particlesassociated with the endoplasmic reticulum of othertissues are much less abundant in muscle.

In the ratio of protein to nucleic acid at least themyofibrillar ribonucleoprotein resembles the micro-somal ribonucleoprotein particles obtained fromother tissues, although its sedimentation behavioursuggests that it is considerably smaller than themicrosomal particles ofcharacteristic sediimentationvelocity which have been reported in the literature(see Tissieres & Watson, 1958, for review). It isnot unreasonable to expect the muscle cell, with itsspecial problem of synthesis of the myofibrillarproteins, to have an intimate association betweenribonucleoprotein and the myofibrils. The ribo-nucleoprotein of the myofibrils may fiulfil a role inthe synthesis of the contractile proteins comparablewith that accredited to the ribonucleoprotein of theendoplasmic reticulum of other cells, and it is hopedto explore these possibilities in future work.

SUMMARY1. A ribonucleoprotein has been isolated as a

minor component of skeletal-muscle myofibrils andcharacterized.

2. Ribonucleic acid of the high-guanine, low-uracil type constitutes about 50% of the nucleo-protein.

3. The bulk of the protein component does notappear to be identical with the known myofibrillarproteins.

4. In muscle homogenates about 60% of thetotal ribonucleic acid in the cell sediments at lowspeeds of centrifuging and most of this nucleic acidis associated in some way with the myofibrils.We wish to express our thanks to Miss Freda Johnson for

her skilled technical assistance, to Mr B. Boon for his helpin the ultracentrifuge experiments and to Dr J. P. Wailerfor a gift ofECTEOLA-cellulose. We are also grateful to theMedical Research Council for a research expense grant (toS.V.P.) which defrayed in part the cost of this investiga-tion.

REFERENCESBain, J. A. & Rusch, H. P. (1944). J. biol. Chem. 153, 659.Beaven, G. H., Holiday, E. R. & Johnson, E. A. (1955).In The Nucleic Acid8, vol. 1, p. 493. Ed. by Chargaff, E.& Davidson, J. N. New York: Academic Press Inc.

Brown, A. H. (1946). Arch. Biochem. 11, 269.Caspersson, T. & Thorell, B. (1942). Acta physiol. scand. 4,

97.Ceriotti, G. (1955). J. biol. Chem. 214, 59.Clavert, J., Mandel, P., Mandel, L. & Jacob, M. (1949).

C.R. Soc. Biol., Paris, 143, 539.Dische, Z. (1930). Mikrochemie, 8, 4.Dounce, A. L. (1955). In The Nucleic Acids, vol. 2, p. 93.

Ed. by Chargaff, E. & Davidson, J. N. New York:Academic Press Inc.

Fiske, C. H. & Subbarow, Y. (1925). J. biol. Chem. 66,375.

Gerendas, M. & Matoltsy, A. G. (1948). Hung. acta phys.1, 124, 128.

Greenstein, J. P. & Thompson, J. W. (1943). J. nat.Cancer Inst. 4, 275.

Hamoir, G. (1951a). Biochem. J. 48, 146.Hamoir, G. (1951 b). Biochem. J. 50, 140.Hasselbach, W. & Schneider, G. (1951). Biochem. Z. 321,

462.Hendler, R. W. (1956). J. biol. Chem. 223, 831.Loftfield, R. B. (1957). Progr. Biophys. Biophysical Chem.

8,347.Magasanik, B. & Chargaff, E. (1951). Biochim. biophys.

Acta, 7, 396.Markham, R. (1955). In Modern Methods of Plant Analysis,

vol. 4, p. 266. Ed. by Paech, K. & Tracey, M. V. Berlin:Springer-Verlag.

Markham, R. & Smith, J. D. (1952). Biochem. J. 52, 558,565.

Mihalyi, E., Brodley, D. F. & Knoller, M. I. (1957).J. Amer. chem. Soc. 79, 6387.

Mihalyi, E., Laki, K. & Knoller, M. I. (1957). Arch.Biochem. Biophys. 68, 130.

Needham, D. M. & Cawkwell, J. M. (1957). Biochem. J. 65,540.

Perry, S. V. (1952). Biochim. biophys. Acta, 8, 499.Perry, S. V. (1953). Biochem. J. 55, 114.Perry, S. V. & Corsi, A. (1958). Biochem. J. 68, 5.Perry, S. V. & Grey, T. C. (1956). Biochem. J. 64, 184.Perry, S. V. & Zydowo, M. (1958). Proc. 4th Int. Congr.

Biochem., Vienna, p. 83.Perry, S. V. & Zydowo, M. (1959). Biochem. J. 71,

220.Peterson, E. A. & Sober, H. A. (1956). J. Amer. chem. Soc.

78,751.Porter, K. R. & Palade, G. E. (1957). J. biophys. biochem.

Cytol. 3, 269.Roth, J S. & Milstein, S. W. (1952). J. biol. Chem. 196,

489.Schmidt, C. G. & Schlieff, H. (1956). Z. ges. exp. Med. 127,

53.Sheng, P-K. & Tsao, T.-C. (1954). Acta physiol. sinica, 19,

203.Szent-Gy6rgyi, A. G., Mazia, D. & Szent-Gyorgyi, A.

(1955). Biochim. biophys. Acta, 16, 339.Tarr, H. L. A. (1958). Annu. Rev. Biochem. 27, 223.Tissibres, A. & Watson, J. D. (1958). Nature, Lond., 182,

778.Volkin, E. & Cohn, W. E. (1953). J. biol. Chem. 205, 767.Warburg, 0. & Christian, W. (1941-42). Biochem. Z. 310,

384.Woodward, G. E. (1944). J. biol. Chem. 156, 143.Wyatt, G. R. (1951). Biochem. J. 48, 584.