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Biochimica et Biophysica Acta, 415 (1975) 311-333 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 85151 MEMBRANE TRANSPORT DURING DEVELOPMENT IN ANIMALS* ANTHONY MARTONOSI Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Mo. 63104 (U.S.A.) (Received April 29th, 1975) CONTENTS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 II. Sarcoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 A. Developmental changes in the Ca 2+ transport activity, composition and structure of sarcoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . 312 B. Mechanism of regulation of Ca 2+ transport during development ......... 316 1. Synthesis of Ca 2+ transport ATPase or its precurser . . . . . . . . . . . . . 316 2. Incorporation of the transport ATPase into the membrane . . . . . . . . . . 318 3. Modification of ATPase enzyme . . . . . . . . . . . . . . . . . . . . . 318 C. Developmental changes in the phospholipid composition of sarcoplasmic reticulum. 320 D. Developmental changes in the physical properties of membrane phospholipids during development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322 E. Effect of developmental changes in fatty acid composition on the ATPase activity and passive Ca 2+ permeability . . . . . . . . . . . . . . . . . . . . . . . . . . 323 F. Ca 2+ binding proteins of sarcoplasmic reticulum in developing chick muscles .... 324 III. Developmental changes in (Na ÷ K+)-ATPase activity . . . . . . . . . . . . . . . . 325 IV. Sugar transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 V. Amino acid transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328 VI. Transport of nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329 VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330 Acknowledgements. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331 * The original work described in this review was supported by the National Institutes of Health, United States Public Health Service (NS 07749 and AM 18117), the National Science Foun- dation, and the Missouri Heart Association. ** Abbrevation: EGTA, ethyleneglycol bis(~t-aminoethylether)-N, N'- tetraacetic acid.

Membrane transport during development in animals

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Biochimica et Biophysica Acta, 415 (1975) 311-333 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands

BBA 85151

M E M B R A N E T R A N S P O R T D U R I N G D E V E L O P M E N T I N A N I M A L S *

ANTHONY MARTONOSI

Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Mo. 63104 (U.S.A.)

(Received April 29th, 1975)

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

II. Sarcoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

A. Developmental changes in the Ca 2+ transport activity, composition and structure of sarcoplasmic reticulum . . . . . . . . . . . . . . . . . . . . . . . . . . . 312

B. Mechanism of regulation of Ca 2+ transport during development . . . . . . . . . 316

1. Synthesis of Ca 2+ transport ATPase or its precurser . . . . . . . . . . . . . 316

2. Incorporation of the transport ATPase into the membrane . . . . . . . . . . 318

3. Modification of ATPase enzyme . . . . . . . . . . . . . . . . . . . . . 318

C. Developmental changes in the phospholipid composition of sarcoplasmic reticulum. 320

D. Developmental changes in the physical properties of membrane phospholipids during development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 322

E. Effect of developmental changes in fatty acid composition on the ATPase activity and passive Ca 2+ permeability . . . . . . . . . . . . . . . . . . . . . . . . . . 323

F. Ca 2+ binding proteins of sarcoplasmic reticulum in developing chick muscles . . . . 324

III. Developmental changes in (Na ÷ K+)-ATPase activity . . . . . . . . . . . . . . . . 325

IV. Sugar transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

V. Amino acid transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328

VI. Transport of nucleosides . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

VII. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

* The original work described in this review was supported by the National Institutes of Health, United States Public Health Service (NS 07749 and AM 18117), the National Science Foun- dation, and the Missouri Heart Association.

** Abbrevation: EGTA, ethyleneglycol bis(~t-aminoethylether)-N, N'- tetraacetic acid.

312

I. INTRODUCTION

Although there are many interesting observations on developmental changes in the activity of sugar, amino acid, nucleoside and ion transport systems, the mole- cular basis of these changes is largely unknown and their implications upon the biogenesis of membranes, the mechanism of transport, or the regulation of gene expression during development are essentially unrealized. The primitive state of our knowledge imposes serious limitations upon the factual scope of the review.

The principal reason for this is that, with few exceptions, the molecular struc- ture of transport pumps and the mechanism of transport are not known in sufficient detail to permit description of developmental changes in transport activity in terms of the underlying fundamental processes.

The only system where some advance has been made in this direction is the sarcoplasmic reticulum, where massive developmental changes in Ca 2+ transport activity were definitively connected with changes in the concentration of a Ca 2+- activated ATPase enzyme in the membrane [1-3]. Discussion of this system will constitute the main topic of the review. The closely related (Na + K+)-ATPase and the developmental changes in sugar, amino acid and nucleoside transport will be only briefly mentioned.

II. SARCOPLASMIC RETICULUM

IIA. Developmental changes in the Ca z+ transport activity, composition and structure of sareoplasmic retieulum

Sarcoplasmic reticulum is a highly differentiated network of intracellular membrane tubules and cisternae, which is present in varying amounts in most muscle cells [4]. Its primary function is the regulation of sarcoplasmic Ca/+ concentration which, in turn, defines the contractile state of myofibrils and the rate of several metabolic processes [5]. Sarcoplasmic reticulum membranes are able to accumulate Ca 2+ against large electrochemical gradients and in this process a membrane bound (Mg 2+ + Ca2+)-activated ATPase plays a central role. The Ca 2+ transport function survives the disruption of muscle cell and can be readily demonstrated in suspensions of microsomal vesicles* obtained from homogenized muscle if ATP is provided as energy source and Mg 2+ as an activator of the transport ATPase [6].

During early embryonic development skeletal muscles perform little work and microsomes isolated from them are essentially devoid of Ca 2+ transport activity [1-3, 7-10]. The increase in the rate of Ca2+-sensitive ATP hydrolysis and active Ca 2+ transport in isolated chicken microsomes at the time of hatching (Fig. 1)

* Skeletal muscle microsomes are usually denoted as fragmented sarcoplasmic reticulum (FSR), although the preparations usually contain varying amounts of mitochondrial and surface membrane elements as contaminants.

313

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Hatching

, -

_ . , , ' _ . , ~ ^ . - ~"

]O 20 30 40 50 60 70

Days of DeveloPment

2~

I

Fig. 1. Developmental changes in ATPase activity, Ca 2+ transport and phosphoprotein concentration. For technical details see ref. 2. ( G - - G ) , Total ATPase activity measured in a medium of 0.1 M KCI, 10 mM imidazole (pH 7.3), 5 mM ATP, 5 mM MgC12, 0.5 mM ethyleneglycol-bis (aminoethyl)- tetra-acetic acid and 0.45 mM CaCI2; ( A - - A ) , Ca 2÷ sensitive ATPase activity. The difference between ATPase activities measured in the above medium with or without 0.45 mM CaCI2 ; ( A - - A ) , concentration of phosphoprotein intermediate measured in a medium of 0.05 M KCI, 5 mM imida- zole, pH 7.3, 0.5 mM [32p]-ATP, 5 mM MgCI2, 0.5 mM EGTA, 0.45 mM CaCI2 and 0.34).6 mg microsomal protein/ml; ( [ ] - - [ ] ) , Ca 2+ uptake was measured as described earlier [2].

coincides with increasing muscular activity. Similar observations were made on sarcoplasmic reticulum fragments isolated from rabbit as well.

Several independent lines of evidence indicate that the increase in Ca 2÷ trans- port activity during development reflects an increase in the concentration of Ca 2+ transporting structures in sarcoplasmic reticulum membranes, rather than a simple increase in enzyme activity due to changes in Km or V.

The hydrolysis of ATP by the Ca 2+ transport ATPase involves a phospho- protein intermediate which has many of the characteristics of an acylphosphate [11-14]. The stability of the acylphosphate bond at slightly acidic pH permitted the covalent labeling of the active site of the enzyme with [32p]ATP [13,15], acetyl [32p]phosphate [15,16] or [a2p]orthophosphate [17]. The maximum amount of covalently bound 32p under optimum conditions approaches 1 mol/mol of enzyme using acetyl [32p] phosphate as substrate [16] while it is usually close to 0.5 mol/mol enzyme with [32p]ATP [18].

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 32p-labeled membranes permitted the identification of the Ca 2+ transport ATPase as a protein of 100 000-dalton mass [15 ].

These observations were applied to sarcoplasmic reticulum membranes isolated from skeletal muscles of developing chicks. The steady-state concentration of phosphoprotein intermediate, used as an indicator of enzyme concentration, increases with development (Fig. 1) roughly parallel with the increase in Ca 2+ transport or Ca2+-sensitive ATPase activity (Fig. 1). The 100000-dalton protein which was

314

previously identified with the Ca 2÷ transport ATPase is a relatively minor component of the membrane at 10-14 days of development but rapidly accumulates around the time of hatching, and in 7-10-day old chicks it constitutes close to 50 ~ of the protein content of the membrane (Fig. 2). Sarcoplasmic reticulum isolated from heart muscle contains less protein in the 100000-dalton band in adult animals but this level is reached earlier in development, in agreement with the early appearance of heart function in chicken embryos [1 ].

Freeze-etch electron microscopy of sarcoplasmic reticulum membranes isolated at 10-14 days of development reveals 75 A intramembranous particles which are more numerous on the outer than on the inner fracture faces (Fig. 3). The density of the particles is about 300-500//zm 2 surface area. During development the density of 75 A intramembranous particles rapidly increases, and 2-3 weeks after hatching it exceeds 4000 particles/#m 2 surface area (Fig. 3).

The diameter of the particles (75 A) assuming spherical shape is consistent with a protein of 100000-dalton mass, and the density of the particles at various stages of development is close to the concentration of ATPase sites in the membrane calculated from the maximum steady-state concentration of phosphoprotein intermediate. These

100, - 80,000 - -

C

Fig. 2. Protein composition of chicken muscle microsomes at various stages of development. A, 14 day; B, 17 day; C, 19 day; D, 23 day; E, 26 day; F, 36 day; G, adult rabbit microsomes. For technical details see ref. 2.

315

Fig. 3. Freeze-etch replicas of sarcoplasmic reticulum from intact muscle. A, 14-day embryo; B, 18-day embryo; C, 7-day posthatched chick; D, 3.5-week posthatched chick. (I) Inner fracture faces; (O), Outer fracture faces. The number of 75 A intramenbramous particles increases with devel- opment on the outer fracture face from 406 (A) to 3853 (D). Theinner fracture faces contain much fewer particles. (Reprinted from ref. 3 with permission from the American Society of Biological Chemists.) x 60000.

316

observations support the probable identity of 75 A particles with the Ca 2+ transport ATPase.

Sarcoplasmic reticulum fragments isolated from adult chicken (Fig. 4) and rabbit [19] also contain surface particles of 40 A diameter which are detectable by negative staining with potassium phosphotungstate. These particles are either absent or difficult to visualize in microsomes isolated from 14-day-old chick embryos (Fig. 4). The density of 40 A surface particles also rapidly increases during develop- ment roughly parallel to the concentration of Ca 2+ transport ATPase. Although they are probably related to the ATPase enzyme, their density in adult animals (15000- 25 000 particles per/~m 2) is much greater than that of the intramembranous particles, prompting further investigation.

The parallel rise in the rate of Ca 2+ transport, the steady-state concentration of phosphoprotein intermediate, the concentration of ATPase enzyme protein and the density of 75 A intramembranous particles during development form the basis of the conclusion that the regulation of Ca 2+ transport activity in sarcoplasmic reticulum is achieved primarily by regulating the concentration of the Ca 2÷ transport system in the membrane.

l iB. Mechanism of regulation of Ca 2+ transport during development The mechanism of regulation of Ca 2+ transport requires the consideration of

the following processes: (a) gene expression which defines the rate of synthesis of Ca 2+ transport ATPase or its precursor; (b) incorporation of the ATPase protein into the membranes either by insertion into a preformed lipid bilayer or by assembly from lipoprotein complexes; (c) possible modification of the ATPase enzyme before or after its incorporation into the lipid phase of the membrane; (d) addition of other membrane components (phospholipids, proteins, etc.) which are required for the function of the Ca 2+ transport ATPase.

Very little is known about any of these processes, but some speculation about future possibilities may be useful.

IIB-1. Synthesis of Ca 2+ transport ATPase or its precursor. The process is probably governed by the same principles as gene expression in other developing systems [20]. The concentration of Ca 2+ transport ATPase in sarcoplasmic reticulum membranes increases 8-10 fold between 10 and 21 days of development [2,3] and in adult muscles may amount to 5-10 mg/g wet muscle weight. Therefore the mechanism of synthesis of the Ca 2+ transport ATPase may be studied in vitro using isolated polysome fractions in experiments similar to those described by Schimke and his collaborators [21] employing purified antibodies against the Ca 2+ transport ATPase [22,23]. These experiments may eventually form the basis of gene quantitation using a purified ATPase messenger RNA [24].

In vitro synthesis of a membrane protein may not only help to understand the regulation of gene expression during development but could provide some infor- mation about the mode of incorporation of nascent polypeptide chains into preformed membrane bilayers.

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The sarcoplasmic C a 2+ concentration is influenced by the sarcoplasmic reti- culum, the Ca 2+ flux through the surface membrane and the Ca 2+ transport function of mitochondria. The surface membranes of cardiac and presumably of skeletal muscles become more permeable to K + as development proceeds [41,42], and it is likely that this permeability change extends to Ca 2+ and other ions as well. An increase in irttracellular Ca 2+ concentration caused by the influx of Ca 2+ through the excitable surface membrane during depolarization could represent the primary stimulus for the increased synthesis of Ca 2+ transport ATPase during development.

IIB-2. Incorporation of the transport ATPase into the membrane. A striking feature of developing sarcoplasmic reticulum membranes is that the phospholipid-rich membranes of 10-14 day old embryos are converted during the next two weeks into protein-rich membranes by a massive increase in the concentration of the Ca 2+ transport ATPase (Fig. 2). These changes are also reflected in a progressive increase in the equilibrium density of the membranes analysed by isopyknic sucrose gradient centrifugation [2], in a corresponding change in the phospholipid/protein ratio (Fig. 5) and in a 10-fold increase in the density of intramembranous particles observed by freeze-etch electron microscopy [3] (Fig. 3). As the yield of microsomal phospho- lipids per g muscle changes only slightly during the same period of development, the results suggest incorporation of Ca 2+ transport ATPase into preexisting phospholipid membranes. Data on the turnover of membrane phospholipids during development could provide additional test of this conclusion.

IIB-3. Modification of ATPase enzyme. The Ca 2+ transport ATPase of chicken sarcoplasmic reticulum represents only a small portion of the total ATPase activity

.E

~- 1.0

a-

0,5

o

o g_

~ 10

I I I / /

o

o t3 f

t ~ i IO 20 30 4~0 */ 65

Days of Development

Fig. 5. Changes in the phospholipid content of chicken microsomes during development. A, Lipid phosphate content of microsomes (0) and of Folch extracts (O); B, Phospholipid composition. O, Lecithin; [] phosphatidylethanolamine; A, phospbatidylserine; +, sphingomyelin with phospba- tidylinositol. Reprinted from ref. l with permission of the publisher.

319

assayed in the presence of 5 mM MgC12 and about 10 - s M Ca 2+ (Fig. 1). The Ca 2+ insensitive portion of ATPase activity rises during development much faster than the Ca 2+ transport ATPase, reaches a maximum around 18 days of development and then declines to a lower steady level, while the Ca 2+ transport ATPase continues to increase.

The Ca2+-insensitive ATPase may perform a metabolic function entirely un- related to Ca 2+ transport, or it may represent a Ca2+-insensitive precursor of the Ca 2+ transport ATPase. Chicken microsomes isolated at various stages of develop- ment were phosphorylated with [a2p]ATP and the proteins containing covalently bound 32p were separated bysodium dodecyl sulfate-polyacrylamide gel electrophoresis (Fig. 6). At early stages of development (10-14 days) two radioactive protein bands were observed with molecular weights of 100000 (Band I) and 80000 (Band II). In microsomes of 14-day old embryos the 80000 molecular weight band contained

60-90 ~ of the total protein bound radioactivity. During further development the radioactivity of Band II slightly decreased while that of Band I increased, and after hatching most of the protein bound 32p was associated with Band I.

c

'~ 300 200 lOO

8 12oo

200. ~

150]'- I A 14day (~ "1 ,ooF 50

23day (~)

800 4O0

Top 10 20 Fractions

1200

800

400

Fig. 6. Separation of microsomal proteins phosphorylatexi with [a2P]ATP. Microsomes were in- cubated with [32p]ATP in a medium containing buffer, KCI, 5 mM NaN3 and 5 mM MgCl2 with 10 -s M Ca 2+ (@) or 5 mM CaCl2 (O) as activators as described earlier. The reaction was stopped with trichloroacetic acid and the proteins were fractionated by sodium dodccyl sulfate-polyacryl- amide gel electrophoresis [15].

320

The phosphorylation of Band II requires Mg 2÷ and is not observed with 5 mM CaClz as the principal divalent cation. Band I is phosphorylated under both con- ditions.

The 100000 dalton band contains the Ca 2÷ transport ATPase. The identity of the 80000 dalton phosphate acceptor protein requires further consideration. The possibility that it may represent contamination or a product of proteolysis was investigated. Isolated mitochondria do not contain this component, and 5mM NaN3 has no effect on the labeling. Therefore it is unlikely that the 80000 dalton protein would arise from contamination with mitochondria. The molecular weight of the phosphate acceptor subunit of (Na + + K+)-ATPase is much greater than 80000 [25]. Contribution by proteolysis is also unlikely since phenylmethyl sulfonylfluoride was included in all isolation and assay media and the 80000 dalton band was observed in fresh or aged microsomes in roughly similar amounts. The usual proteolytic fragments of the Ca 2+ transport ATPase have much lower molecular weights (50000-60000) [26] than the 80000 dalton acceptor.

Upon fractionation of crude microsomal preparations by isopyknic sucrose gradient centrifugation the relative amount of radioactivity in band I and band II remained constant in membrane fractions collected from different regions of the gradient. These observations suggest that the two acceptot proteins are probably components of the same membrane fraction. The association of phosphorylase with sarcoplasmic reticulum membranes has been frequently suggested. Although the molecular weight of the Band II protein is somewhat smaller than that of phosphoryl- ase, further work is required to exclude conclusively their identity.

The decline in the radioactivity of Band II parallel with the massive increase in Band I during development raises the possibility of a precursor-product relationship between the two proteins. If this is the case, the Ca 2+ transport ATPase may arise by covalent chemical modification from the precursor protein. It is interesting in this regard that the phosphorylation of Band II and Band I have different Ca 2 + require- ments. Alternatively Band II may represent an entirely independent enzyme which operates through an acylphosphate intermediate.

Decision between these alternatives may be made by isolation of the Band II protein and analysis of its immunochemical characteristics and fingerprint in com- parison with the Ca 2+ transport ATPase.

IIC. Developmental changes in the phospholipid composition of sarcoplasmic reticulum The Ca 2+ transport of sarcoplasmic reticulum requires membrane phospho-

lipids in at least two phases of the process: (a) The (Mg 2÷ + Ca2+)-activated ATP hydrolysis is inhibited by removal of membrane phospholipids with a variety of phospholipases [18,27-29], and the activity can be restored with micellar dispersions of synthetic and natural phospholipids [27,28]. The phospholipid requirement of ATPase activity was connected with the hydrolysis of phosphoprotein intermediate [13,29]. (b) Phospholipids are also required for maintaining the permeability char-

321

acteristics of sarcoplasmic reticulum membrane, which is important for the retention of accumulated calcium [23,30-32].

It was of interest, therefore, to determine the developmental changes in the lipid composition of sarcoplasmic reticulum membranes, and to evaluate the contri- bution of these changes to the ATPase activity and Ca 2+ transport.

The phospholipid/protein ratio of isolated sarcoplasmic reticulum fragments decreases during development owing primarily to an increase in the concentration of Ca 2÷ transport ATPase (Fig. 5). During this time there is relatively little change in the contribution of phosphatidylcholine, phosphatidylethanolamine, phosphatidyl- serine, sphingomyelin and phosphatidylinositides to the phospholipid content.

There are marked changes, however, in the fatty acid composition of the mem- brane lipids, namely a decrease in palmitate and increase in linoleate content with smaller changes in other fatty acids (Fig. 7A). The net result of the developmental changes in fatty acid composition is that the early embryonic membranes containing primarily saturated fatty acids (Fig. 7B) are converted by the 30th day of develop- ment into Ca2+-transporting structures in which about 6 0 ~ of the fatty acids are unsaturated. The accumulation of unsaturated fatty acids at the expense of palmitate occurs with an increase in average chain length. This may be of importance in

40

30

20

10

010

50

25

I I I { A

20 30 40 65

14 19 26 33 42

Days of Development

Fig. 7. Changes in the fatty acid composition of microsomal phospholipids during development. A: O, palmitate; II, stearate; [], oleate; O, linoleate; A, arachidonate; +, myristoleate; ~ , palmitoleate. B: II, saturated fatty acids (stearate + palmitate); hatched area, short-chain (C-14 and C-16) unsaturated fatty acids; ~, long-chain unsaturated fatty acids (oleate, linoleate, arachidonate). Reprinted from ref. 1 with permission from the publisher.

322

maintaining the proper fluidity of the lipid phase of the membrane at physiological temperature.

liD. Developmental changes in the physical properties of membrane phospholipids during development

Diferential scanning calorimetry of dry sarcoplasmic reticulum membranes isolated after 14 days of development shows two major endothermic transitions at 15-22 and 35 °C, respectively. There is relatively little change in the temperature or magnitude of these transitions up to 37 days of development when the Ca 2+ transport activity is maximal (Fig. 8). The slight change in the temperature of the lower

m

b , , T j i

~ 5 ~ 2 a ~

I I I I I -20 " 0 20 40 60 80 100

° C

Fig. 8. Developmental changes in the thermal capacity of sarcoplasmic reticulum membranes. For technical details see ref. 33. A, 14-day embryo; B, 18-19 day embryo; C, 5-day-old chick; D, 16-day- old chick; E, 4-month-old chicken.

transition to 12 °C in 4-month-old chicken is not accompanied by detectable changes in Ca 2+ transport, and may arise from dietary changes in fatty acid composition.

Dry, extracted microsomal phospholipids also show two endothermic tran- sitions at all stages of development. These occur at somewhat lower temperatures than in intact membranes, and the transition temperatures slightly decrease with development (Fig. 9).

The presence of two transitions in both native membranes and extracted phospholipids may reflect either a mixture of different types of membranes in the preparation or a phase separation of lipids within the sarcoplasmic reticulum mem-

323

~ 0 <,,,!, " I -

,.z~

l a . ~2

-20 0 20 40 60 80 100 ° C

Fig. 9. Thermal analysis of extracted microsomal phospholipids at different stages of development. The lipids were extracted and concentrated to 1-2 ml on Buechler Evapomix under vacuum [2]. 1 ml benzene was added and evaporation continued on Evapomix until a dry powder was obtained and then on freeze-dryer overnight. The samples were scanned between - - 40 and -k 100 °C at a sensitivity of 0.02 mcal/s per inch at a heating rate of 2 °C/min. The figure contains traces of the second scan. A, 14-day embryo (2.4 mg); B, 17-day embryo (2.4 mg); C, 1-day chick (3.0 mg); D, 5-day chick (2.9 mg); E, 15-day chick (3.9 mg); F, 50-day chick (4.5 mg).

brane. Comparable preparations obtained from rabbit muscle displayed ovly one

major transition [33]. The temperatures of the main transitions are shifted to or below 0 °C in the presence of 10--15 ~ cf water in native membranes and in isolated phospholipids. Although Ca z+ and Mg z÷ markedly increase the transition tempera-

ture of sarcoplasmic reticulum membrane lipids, at the temperatures customarily used for the assay of ATPase activity and Ca z÷ transport (25-37 °C) the microsomal phospholipids are expected to be in the liquid crystalline state with relatively little

change in physical properties during development.

liE. Effect of developmental changes in fatty acid composition on the ATPase activity and passive Ca z+ permeability

The question arises of whether the observed developmental changes in Ca z÷ transport reflect the influence of the fatty acid composition of membrane phos- pholipids upon the ATPase activity and passive Ca z+ permeabihty of the membrane. Such effects, if they exist, must be relatively small.

324

The observed changes in ATPase activity are very nearly proportional to the concentration of ATPase enzyme in the membrane, although the phospholipid environment at early and late stages of development is rather different.

The passive Ca 2+ permeability of the membrane increases only slightly between 17 and 23 days of development (Fig. 10) and even in fully developed chicken remains

10.0

._~ -4J

~ 1.0 - -

0.1 - -

1 1 1 1 1 5

I I I I I 10 15 20 25 30

Incubation time, minutes

Fig. 10. The passive Ca 2+ permeability of chicken muscle microsomes during development. The Ca 2+ permeability was measured by Millipore filtration as described earlier (ref. 32). 0, 17 day; O, 23 day, D, 36 day; ram, 81 day; +, adult rabbit.

lower than the permeability of similar membrane fractions obtained from adult rabbits. The passive Ca 2+ permeability of lipid bilayers increases several orders of magnitude upon the incorporation of Ca 2 + transport ATPase [32]. The difference in the passive Ca 2+ permeability of adult chicken as compared with rabbit sarcoplasmic reticulum may be related to the observed differences in the density of Ca 2+ transport sites [2,32].

The absence of a major influence of the developmental changes in fatty acid composition upon the permeability and enzymatic function of the membrane is presumably an indication of the balance between chain length and unsaturation of fatty acids which maintains the fluidity of the bilayer reasonably constant. This conclusion is in harmony with the thermal analysis data that show only slight changes in the transition temperature of membrane lipids with development.

IIF. Ca 2+ binding proteins of sarcoplasmic retieulum in developing chick muscles

Sarcoplasmic reticulum membranes of rabbit [34] and chicken [35] contain two proteins of about 50000 and 60000 molecular weight (C1 and C2 proteins) which are readily released from the membrane by treatment with 1 mM EDTA [34] or dilute

325

deoxycholate [36]. MacLennan discovered that these proteins bind Ca 2+ with high affinity [37 ] and suggested that they may play a role in the binding of accumulated Ca 2+ within the vesicles. As the intravesicular localisation of Ca2+-binding proteins is open to doubt [34,38,39 ], their role in Ca 2+ transport is also uncertain.

The concentration of C1 and C2 proteins in sarcoplasmic reticulum membranes as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is high already at 14 days of development and changes only slightly during the next 2-3 weeks, when massive increase in Ca 2+ transport and in the concentration of Ca 2+ transport ATPase are observed (Fig. 2). The ratio of ATPase/C1 + C2 proteins changes from about 0.3-0.4 at 14 days to 5-6 at 37-40 days of development, largely as a result of changes in the concentration of Ca 2+ transport ATPase.

If the C1 and C2 proteins represent genuine components of sarcoplasmic reticulum, their synthesis and binding to the membrane occurs before the incorpo- ration of Ca 2+ transport ATPase. Alternatively, the possibility must be considered that at least a portion of the C1 and C2 proteins may represent a contamination in the preparations of both embryonic and adult microsomes. Mitochondrial fragments are known to be present in sarcoplasmic reticulum preparations. The C1 and C2 proteins are similar in several respects to the two large subunits of mitochondrial ATPase (F 0, although the reported amino acid compositions of F1 subunits [40] and the C1 and C2 protein do not quite agree.

III. DEVELOPMENTAL CHANGES IN (Na ÷ 4- K+)-ATPase ACTIVITY

Regulation of (Na + + K+)-ATPase activity during development and in response to changing conditions during adult life is of fundamental importance in the mainte- nance of intracellular Na + and K + concentrations in all cells. It is expected that cells are capable of maintaining ion gradients by adjusting their pumping capacity to changes in Na + influx or extracellular K + concentrations. This may be of particular significance in excitable tissues such as muscle or nerve.

In developing chicks the (Na + ÷ K+)-ATPase activity of cardiac and skeletal muscles increases with age (Fig. 11). In cardiac muscles the maximum rate of (Na + ÷ K+)-ATPase activity is reached around the time of hatching, while in skeletal muscles it continues to rise at least until the 30th day of development [41]. The increase in the activity of (Na + + K+)-ATPase parallels the increase in the K + per- meability of the cell membranes with development [42]. Even though the specific activity of the ( N a + + K+)-ATPase is low in early embryonic heart it is sufficient to maintain the low intracellular Na + and high K + concentrations, because the passive K + permeability is also low. In 16-day embryos the Na + pumping rate was estimated to be about 18 - 10 -12 tool Na+/cm z per s which, assuming a turnover rate of 20 s-1, would give about 1900 pumping sites per/~m z surface area [41]. As the principal kinetic characteristics of the ATPase in cultured heart cells are constant under a variety of conditions [43 ], it is assumed that the developmental changes in (Na + + K+) -

326 RELATIVE

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Fig. 11. (Na ÷ + K÷)-ATPase activity of cardiac and skeletal muscle of the chick as a function of development. For details see ref. 41. Closed symbols give relative activity with respect to the activity at day 16 and open symbols give absolute activity. (3, e , heart; ~ , El, skeletal muscle.

ATPase activity reflect the changes in the concentration of (Na + -- K+)-ATPase in the membrane. Essentially similar observations were also made by Klein [44] on embryonic chick heart, and by Zaheer et al. [45] on embryonic chick brain. In rat brain [46] the increase in (Na++K÷)-ATPase occurs at birth, and adult levels are reached by the 12th postnatal day.

Information is beginning to emerge concerning possible mechanisms of regu- lation of the ( N a ÷ ÷ K+)-ATPase activity in cells.

In guinea pigs kept on a K÷-free diet the plasma K ÷ level decreases, accom- panied by an increase in the (Na ÷ + K÷)-activated ATPase of heart cells [47]. Increase was also observed by Chan and Sanslone [48] in the (Na ÷ + K÷)-ATPase activity of erythrocytes of potassium depleted rats.

327

Cells appear to match their (Na + 6- K +) transport capacity to the intracellular concentration o fNa + [49,50]. The increased passive Na + leakage through erythrocyte membranes in hereditary spherocytosis is accompanied by increased pumping activity [51]. After partial inhibition of (Na + 6- K +) transport in cultured HeLa cells by ouabain, the cell generates new ouabain-binding sites and the transport activity is completely restored in about 3 h [52]. The recovery is inhibited by cycloheximide, indicating a requirement for protein synthesis. Growth of cells in media of low K + concentration also enhances the concentration of the transport enzyme, in agreement with the suggestion that the synthesis of (Na + 6- K+)-ATPase is regulated by the electrolyte content of the cell [52,53]. The mechanism of regulation may differ in various tissues, as K + loading apparently increases the concentration of (Na + 6- K+) - ATPase in kidney [54].

Although various regulatory parameter s are stressed by these experiments, the possibility exists that in each case more than one factor contributes to the observed changes in (Na + 6- K+)-ATPase activity.

The mechanism of adjustment apparently fails in low K + (LK) sheep where the K + content of erythrocytes is only 14-16 mM as compared with 70-90 mM in normal (HK) sheep. Correspondingly the ( N a + + K+)-ATPase activity is 4-8 times greater in HK than in LK cells [55]. This difference in transport activity was previously explained by the difference in the number of transport sites measured by [3H]ouabain binding [55].

It appears, however, that the low rate of active transport in LK cells results largely from competitive inhibition of internal Na ÷ sites by K + for which LK 'cells have an unusually high affinity [57-59]. Interestingly, LK cells are also more permeable to K ÷ than HK cells.

Treatment of LK cells with antiserum prepared against other LK cells increases the rate of K ÷ transport [60] by increasing the relative affinity of internal binding sites for Na ÷ [57-59]. Although the rate of ouabain binding increases after attach- ment of antibodies [56], an earlier suggestion that antibody increases the number of oubain binding sites [61] was not confirmed.

In prenatal or newly born LK lambs the cation composition and transport activity of erythrocytes is similar to cells from HK sheep. During the next 60 days of development there is a conversion to the LK phenotype, either by replacement of fetal with adult cells or by a conversion of pumping sites into leak sites on cells remaining in circulation [62-66]. The lability of the transport sites in LK cells is apparently determined by a single genetic locus [67].

These examples emphasize the possible role of feedback regulation by ions in the synthesis or activation of (Na ÷ 6- K+)-ATPase sites in membranes. Examples of this type of regulation are also encountered in amino acid transport (see below).

328

IV. SUGAR TRANSPORT

Mediated transport of sugars is a common feature of all animal cells, although the rate of the process varies widely depending on species, cell types and age [68]. The rate of glucose transport by human erythrocytes is about 250-300 times faster than in erythroctes of nonprimates (guinea pig, rabbit, sheep) although the various systems are similar in stereospecificity and Kin, and all exhibit countertransport.

Widdas observed [69] that fetal elythrocytes of nonprimates transport glucose at a fast rate like human cells, but during postnatal development the transport rate rapidly declines, presumably by exchange of fetal with adult cells. As the biochemical nature of glucose carrier is poorly defined [70,71] it is difficult to tell whether this represents a change in the activity or in the concentration of carrier sites.

Changes in glucose metabolism during development have been observed in many cell types of several species, but it is generally difficult to separate the transport component in these observations from the indirect effects of metabolism on sugar uptake.

In 5-day-old chick embryo hearts the uptake of glucose is rapid and the relation- ship between the rate of uptake and the external glucose concentration suggests free diffusion [72,73]. At this early phase of development insulin has no effect on the glucose uptake [74] and the solbitol space is large, indicating that the surface mem- brane is freely permeable to various sugars. In embryonic hearts 7 days or older the rate of glucose uptake is progressively reduced together with a decrease in sorbitol space, and the uptake of glucose becomes a saturable process that is sensitive to insulin [72-74]. These observations are consistent with the appearance of a glucose carrier in chick heart at around the 7th day of embryonic development.

The ability to accumulate glucose and galactose appears in chicken intestinal slices only on the day of hatching, and reaches a plateau 2 days later [75]. In guinea pig intestine the absorption of a-methylglucoside shows a marked increase before birth and then achieves a somewhat lower adult level 2 weeks later [76]. Similar observations were also made in the rabbit [77]. In most mammals the intestinal sucrase and isomaltase develop after birth, at the time of weaning. An exception is man, where adult activities are reached before birth [78]. The transport rate of 3-0- methylglucose across the blood-brain barrier is one-fourth of the adult rate in new- born rat and reaches maximum only at the 20th postnatal day. Very little is known about the mechanism of this process [79].

V. AMINO ACID TRANSPORT

In guinea pigs during the first postnatal day the transport of glycine and 1-amino-cyclopentanecarboxylic acid into liver and to a lesser extent into muscle markedly increases [80], approaching adult levels [81,82]. The increase in transport activity is probably initiated by the rapid decrease in plasma glycine concentration

329

during the first few hours after birth [80]. The transport system under this type of control is apparently the Na+-dependent system A [83]. The mechanism of the regulation of amino acid transport was investigated in a series of elegant experiments by Guidotti and his collaborators [84-87]. In isolated 5-day heart or in suspensions of cardiac muscle cells exposed to amino acid-free Krebs-Ringer bicarbonate for a few hours there is a sharp increase in the rate of amino acid uptake, with a specificity pattern similar to that of system A. The enhancement of amino acid transport is prevented by puromycin or by the inclusion of system A substrates into the incubation medium. Other amino acids were ineffective. Tentative evidence was obtained that the regulation of transport activity by amino acids occurs at the transcriptional level [85], where insulin also exerts one of its effects on amino acid transport [87].

Similar mechanisms may be also operative in skeletal muscle, kidney [88,89] and placenta [82]. Increase in aminoisobutyric acid and cycloleucine transport was also observed after viral transformation of several animal cell lines [90,91].

During maturation of rabbit reticulocytes some amino acid transport systems are retained unmodified, while others are gradually lost. The repression of alanine transport during maturation occurs faster than that of lysine [92], suggesting the involvement of different transport systems. Rabbit reticulocytes accumulate glycine against a concentration gradient, while mature rabbit erythrocytes no longer possess any concentrative ability [93].

These differences in the amino acid transport characteristics of reticulocytes and erythrocytes [94,95] are reminiscent of the repression of sugar transport in non- primate erythrocytes during early postnatal development [69].

The enzymatic basis of amino acid transport is largely unknown. The possible role of membrane bound 2-glutamyl transpeptidase in amino acid transport was suggested by Meister [96,97]. Fetal kidney has little or no 2-glutamyl transpeptidase activity [98,100]; the activity increases during development and adult levels are reached in 7-week-old rats. In contrast, fetal and neonatal liver, lung and brain possess much higher transpeptidase activities than adult organs, and the activity decreases during development [98,101 ].

VI. TRANSPORT OF NUCLEOSIDES

Tissue culture studies offer interesting insight into the relationship of transport processes to cell cycle [102] and differentiation [10,102].

The entry of uridine into Novikoff rat hepatoma cells [103] occurs by at least two processes: (a) a saturable facilitated diffusion which is dominant at uridine con- centrations less than 50-100 #M; (b) a nonsaturable process which exhibits low temperature coefficient, a linear dependence on nucleoside concentration and presum- ably represents free diffusion. Similar observations were made on the uptake of adenosine [I04], thymidine [105], guanosine, inosine and cytidine [104].

In synchronized cultures of Chinese hamster or Novikoff cells the rates of

330

transport of uridine, thymidine and other metabolites display characteristic changes in various stages of the cell cycle [106,107], supporting the conclusion that specific systems are involved in the transport of different nucleosides. The increases in transport rates during the cell cycle were prevented by actinomycin, cycloheximide or puromycin [106,107], indicating that these changes require both transcription and translation.

The thymidine and uridine transport are rapidly lost after treatment of the Novikoff cells with actinomycin D or cycloheximide, while the choline and 2-deoxy- glucose transport are unaffected for several hours [107]. The large scale changes in transport rates under conditions of altered protein synthesis are consistent with the suggestion that nucleoside transport is regulated by changes in the concentration of carrier sites in the membrane.

The rate of uptake of uridine and phosphate increases 2- to 4-fold within 10-15 min after adding fresh serum to confluent 3T3 cells [108]. The increase in transport is inhibited by inhibitors of protein synthesis [109,110]. The increase in uridine uptake is specific as serum has no effect on the uptake of 3-O-methyl-D-glucose, amino acids [108], adenosine [111], deoxyadenosine or deoxyguanosine [112]. The increased uridine and phosphate transport after addition of serum coincides with the initiation of cell division [113]. The mechanism of this regulation is unknown.

vii. SUMMARY

This brief and necessarily incomplete survey of available evidence on the development of transport systems in animal cells reveals a primitive state of knowledge full of interesting possibilities for future development.

The assembly of membrane-bound transport systems during embryonic development provides unique opportunities for approaching questions relating to gene expression, the synthesis and insertion of membrane proteins into phospholipid layers, the composition and structure of transport systems and the conditions required for their functioning.

It seems plausible to assume that the growth and differentiation of animal cells is regulated, in part at least, by the rate of transport of metabolites and ions across the cell membranes. Therefore the sequence of the expression of transport systems is likely to have a profound effect on subsequent stages of growth and differentiation. Feedback regulation of the synthesis of transport proteins by changes in the intra- cellular or extracellular concentrations of the transported metabolites or ions [52, 53, 85-87] may be a key element in the regulation of the rate of transport processes during development.

331

ACKNOWLEDGEMENTS

My thanks are due to Dr R. Boland, T. Tillack, A. Boland, T. Chyn, Fr ieda

For t ie r and R. J i lka for part icipat ing in various phases o f the work.

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