THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 249, No. 2, Issue of January 25, PP. 612-623, 1974

Printed in U.S.A.

Developmental Changes in the Composition and

Function of Sarcoplasmic Reticulum*

(Received for publication, June 13, 1973)

RICARDO BOLAND AND ANTHONY MARTONOSI~

From the Department of Biochemistry, St. Louis University School of Medicine, St. Louis, Missouri 63104

THOMAS W. TILLACK

From the Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110

SUMMARY

Developmental changes in the protein, phospholipid, cholesterol, and fatty acid composition of skeletal and cardiac microsomes were correlated with CaZf transport activity in chicken embryos and during the early postnatal period. In skeletal muscle microsomes isolated from leg, superficial pectoralis, or deep pectoralis muscles, the rapid increase in the rate and extent of calcium accumulation around the time of hatching was accompanied by increase in total and CaZi- sensitive ATPase activity, the concentration of phospho- protein intermediate and the amount of transport ATPase enzyme determined by gel electrophoresis. The increased enzyme content of the membrane is reflected in its increased density and a marked decrease in phospholipid to protein ratio with development. The concentration of phosphatidyl- choline, phosphatidylethanolamine, phosphatidylserine, sphingomyelin, and phosphatidylinositol changed relatively little during the first 50 days of development but the appear- ance of calcium transport activity was accompanied by a marked decrease in the palmitate and increase in the linoleate content of membrane phospholipids and a decrease in choles- terol concentration.

These changes were clearly apparent in purified sarco- plasmic reticulum membranes isolated by calcium loading or in less pure membrane fractions obtained by differential or sucrose gradient centrifugation.

Corresponding but much less pronounced changes in the enzymatic activity of cardiac microsomes were observed during development. Although the observations are con- sistent with nearly all possible mechanisms of membrane assembly they may suggest that the phospholipid-rich mem-

branes of embryonic muscle are gradually converted during

* The work at St. Louis University was supported by Research Grants NS 07749 from the National Institutes of Neurological Diseases and Stroke (A. M.) and GB 33867 X from the National Science Foundation (A. M.). The electron microscopic study at Washington University (T. T.) was financed by: Brown &William- son Tobacco Corp.; Larus & Brother Co., Inc.; Liggett & Myers Inc.; Lorillard, a Division of Loews Theatres Inc.; Philip Morris Inc.; R. J. Reynolds Tobacco Co.; United States Tobacco Co.; and Tobacco Associates, Inc. A preliminary report of some of this work was presented at the Cold Spring Harbor Symposium on The Mechanism of Muscle Contraction in June 1972 (1).

$ To whom all correspondence may be directed.

development into calcium-transporting structures by step- wise insertion of the ATPase enzyme and other components of the transport system.

Sarcoplasmic reticulum membranes offer a relatively simple system for the analysis of the requirements of active ion trans- port (2). The transport ATPase constitutes 60 to 70% of the protein content of the membrane (3). Its molecular weight on the basis of polyacrylamide gel electrophoresis is close to 100,000 (3-5). Under appropriate conditions the enzyme protein is separated into several subfragments with molecular weights ranging from 30,000 to 60,000 (3, 6).

The ATPase activity and calcium transport are reversibly in- hibited by removal of membrane lipids with phospholipase C (7, 8). The activity can be restored with micellar dispersions of synthetic or natural phospholipids indicating a spontaneous re- formation of the native lipoprotein structures (7-9). Self-as- sembly of membrane structures from microsomal components solubilized with deoxycholate is also readily demonstrable (10). The morphological and functional characteristics of the recon- stituted membranes are reminiscent of native sarcoplasmic re- ticulum (5, 10).

Developmental changes in the composition (I), function (1, ll-13), and structure of sarcoplasmic reticulum membranes offer a promising insight into the mechanism of intracellular membrane assembly.

During early embryonic development, chicken skeletal mus- cles are largely inactive and the microsomes isolated from them are devoid of calcium transport activity. The appearance of calcium-sensitive ATPase and Ca2+ transport shortly before hatching coincides with increasing muscular activity. Both functions reach a maximum activity in 2-week-old chicks pro- viding readily available material for the study of the assembly of calcium-transporting structures at all stages of completion. As the calcium transport system of sarcoplasmic reticulum is de- pendent upon membrane phospholipids for activity (8), changes in the lipid composition of the membrane may contribute to the developmental changes in ATPase activity and calcium transport.

The subject of the next two reports is an analysis of the pro-

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tein, phospholipid, and fatty acid composition of membrane fractions isolated from skeletal and cardiac muscles of chicks during embryonic and postnatal development. These data are correlated with developmental changes in the calcium transport, ATPase activity, and phosphoprotein concentration of the mem- branes and with their fine structure as derived from freeze-etch electron microscopy.

EXPERIMENTAL PROCEDURES

White Leghorn eggs were incubated in New Brunswick incu- bator shaker (model G 27) at 38” and a relative humidity of 64 to 70%. After hatching the chicks were maintained on water for 2 days followed by a Ralston Purina Chick Startena Medi- cated 20-V 6011 diet ud l&&m. Microsomes were prepared from leg, superficial pectoralis, deep pectoralis, and heart mus- cles obtained during 10 to 120 days of development beginning with the start of incubation. The number of animals used for one microsome preparation was 100 to 142 with lo-day-old embryos, 60 to 70 with 18. to 20-day-old embryos, 20 to 35 with 15- to l&day-old chicks, and 6 to 7 with 42-day-old chicks. The term embryo is used to denote all stages of development before hatching. Except when otherwise noted, the age of chicks is given in days after hatching.

The muscles were excised, cleaned of fat and connective tis- sue, minced, and placed in an ice bath made of distilled water un- til sufficient amount was collected (usually about 1 to 1.5 hours). The muscles were homogenized for 90 s in a Virtis homogenizer or Waring Blendor using 4 volumes of 0.1 M KCl, 10 mM imi- dazole, pH 7.4, and 0.3 M sucrose. The homogenates were centrifuged for 20 min at 8,200 x g in Lourdes 9RA or VRA rotors. The sediment was discarded. The supernatant was filtered through glass wool previously washed with the homoge- nizing medium. The remaining mitochondria were removed by centrifugation for 30 min at 8,200 X g. The microsomes were sedimented from the supernatant at 50,000 X g for 1 hour in Spinco No. 50 or 30 rotors, depending upon the volume.

The microsomal sediment was dispersed in a solution of 0.6 M

KCl, 10 mM imidazole, pH 7.3, and 0.3 M sucrose. After standing for 30 min at 2”, the microsomes were collected by ccntrifugation at 50,000 X g for 1 hour. The sediment was dispersed in 0.3 hf sucrose and centrifuged at 58,000 x g for 30 min. The final sedi- ment was suspended to a protein concentration of 5 to 10 mg per ml in 0.15 or 0.3 M sucrose, and used either for further fractiona- tion or for the various assays.

Two methods were used for further purification of microsomes. Sucrose Density Gradient I”ractionation-Centrifugation was

performed in Spinco SW 25.1 or 39 rotors at 41,000 x g for 18 to 20 hours using a continuous gradient ranging from 0.3 to 1.50 M sucrose concentration in 10 mM histidine, pH 7.3. The micro- some samples were applied in 0.15 or 0.3 M sucrose at the top of the gradient. After centrifugation, the contents of the tubes were collected in five fractions, diluted to 0.3 M sucrose concell- tration, and centrifuged at 58,000 x g for 1 hour. The sedi- mented microsomes were suspended in 0.3 M sucrose, 10 mM imidazole, and used for the various assays. Fraction 1 repre- sents the bottom layer, and Fractions 2 to 5, the progressively lighter fractions.

Discontinuous Sucrose Gradient Centrijugation of Calcium Oxalate-loaded dficrosomes @)-Leg microsome suspension (0.5 ml containing 5 to 10 mg of protein) was layered on the surface of 4 ml of 407, sucrose-10 rnbf imidazole, pH 7.4, solution in an SW 39 tube and centrifuged in SW 39 rotor at 64,000 x g for 2 hours. The supernatant was decanted and mised with 60-ml

solution containing 0.1 M KCl, 10 mM imidazole, 5 mM calcium oxalate, 5 mM MgC12, 5 mM ATP, and 2.5 x 10d4 M 45CaC12 equilibrated at room temperature. In several experiments, Fraction 3 obtained after centrifugation in continuous sucrose density gradient was used directly for the calcium oxalate- loading experiment. After incubation for 8 min at 25” the solu- tion was rapidly cooled, and loaded in 18-ml portions onto the surface of three gradients containing 6 ml of 40% sucrose-10 mM imidazole, and 6 ml of 28% sucrose-10 mM imidazole in SW 25 tubes. After centrifugation at 51,505 X g for 1 hour the calcium oxalate-loaded microsomes sedimented to the bottom of the tube containing most of the 45Ca radioactivity added to the medium. The supernatants were diluted with water and the microsomes were collected by centrifugation.

Both fractions were dispersed in water to the desired protein concentration and subjected to the various assays described be- low.

Samples were taken from crude microsomes, sucrose density gradient-fractionated microsomes, and calcium oxalate-loaded preparations for electron microscopy in thin sections and by the freeze-etch method.

Measurement of Calcium Uptake-Calcium uptake was meas- ured on the day of microsome preparation at 25” in a medium of 0.1 M KCl, 10 mM imidazole, 5 mM MgC12, 5 mM ATP, 5 mM potassium oxalate, and 0.1 mM 45CaC12 at several protein con- centrations ranging from 0.03 to 0.3 mg per ml. Samples were taken for Millipore filtration (14) after 1, 2, 5, and 10 min of incubation. The rate of calcium uptake and the calcium ca- pacity of the microsomes were evaluated on the basis of the values obtained after l- and IO-min incubation, respectively.

ATPase Activity-ATPase activity was measured in three dif- ferent media. Medium A contained 0.1 M KCl, 10 mM imidazole, 5 mM ATP, 5 mM MgC12, 0.5 mM EGTA, and 0.45 mM CaC12. Medium B was identical with A but with CaC12 omitted. Me- dium C contained, in addition to KCl, imidazole, and ATP, 5 mM CaCl* as activator. The reaction was started with the addition of microsomes (0.015 to 0.075 mg per ml) and after 5 to 20 min incubation at 25” was stopped with trichloroacetic acid. Each sample was tested at at least two different protein concentrations. The liberated inorganic phosphate was assayed according to the method of Fiske and SubbaRow (15). The “extra” or “calcium-sensitive” ATl’ase activity is obtained as the difference between the rates measured in Nediums A and B. Only this calcium-sensitive ATl’ase activity is related to the calcium pump.

Jfeasurement of Phosphoprotein Intermediate-The reaction was performed under three different conditions as described earlier (16). The assay system contained 0.05 M KCl, 5 mM imidazole, 0.5 mM [321’]hTl’, 0.3 to 0.6 mg of microsomal protein per ml, and either 5 m&i l\lgClz, 0.5 mM EGTA, and 0.45 mM CaClz (Medium I) or 5 mM NgClz and 0.5 mM EGTA (,\Iedium II) or 5 mM CaClz (biedium III) as activators. Incubation was carried out in ice for 20 s. The reaction was stopped as described earlier (16). In experiments with crude microsomes, the proc- essing of samples involved repeated centrifugation as reported earlier (16). In the experiments with microsomes purified by sucrose density gradient centrifugation or calcium oxalate sedimentation, after two initial centrifugations, a sample was taken for protein determination and the rest of the microsomes were filtered through Nillipore filter. After three to four addi- tional washings with 2% trichloroacctic acid, the filter was dried and counted in toluene-based scintillation fluid using 2,5-di-

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phenyloxazole (PPO) and 1,4-bis[2-(5.phenyloxazolyl)]benzene (POPOP) as scintillators.

Protein was determined by the Lowry (17) method. In most experiments, the method of Loftfield and Eigner (18) was used for measurement of radioactivity.

The protein composition of crude microsomes, and the mem- branes purified by sucrose density gradient centrifugation or calcium osalate loading was analyzed by polyacrylamide gel electrophoresis essentially as described earlier (3). The elec- trophoretic gels were stained with Amido black and an approxi- mate estimate of the relative amounts of the various proteins was obtained by scanning with Joyce-Loebl Chromoscan densitom- eter.

For determination of phospholipids, microsomes were ex- tracted with chloroform-methanol according to the method of Folch et al. (19) and separated by thin layer chromatography on Silica Gel G using chloroform-methanol-concentrated NH,OH (65:25:4, v/v) as solvent. For two-dimensional separation the solvent systems were : chloroform-methanol-concentrated NH,OH (65:35:5, v/v) and chloroform-acetone-methanol-acetic acid-Hz0 (50 :20 : 10 : 10 : 5, v/v). Normally the phospholipids were detected by exposure to iodine vapors. In some cases, Zinzadze’s reagent (20) or ninhydrin was also used.

For determination of fatty acid composition the total lipid extract of microsomes was evaporated to dryness and subjected to transesterification in methanol-HS04 (95:5, v/v) under nitrogen for 90 min at 100” (21,22). The resulting methyl esters were extracted and concentrated according to the method of Keenan and Morre (23). Gas chromatography was performed on a Barber-Colman series 5000 instrument equipped with an electron capture detector using a glass column (6 feet x 3 mm diameter) packed with 10% EGSS-X on Chromosorb Q (100 to 120 mesh) obtained from Applied Science Laboratory, State College, Pa. The flow rate of argon (Chemetron Corp., Chicago) was 40 ml per min. The carrier gas was dehydrated by passage through a molecular sieve column (Applied Science Laboratories). Column temperature: 180”. Detector temperature: 270”.

The methyl esters of fatty acids were identified by two meth- ods. (a) Comparison of their retention time with those of known fatty acid methyl esters. (b) Analysis of their mass spectra using an LKB model 9000 gas chromatograph-mass spectrometer equipped with a Becker-Ryhage molecule sepa- rator.

For all fatty acid methyl esters except one earlier identified (1) as myristoleate (14: l), the mass spectra were identical with those of aut,hentic fatty acid methyl esters. The compound with the retention time of methyl-myristoleate yielded a mass spectrum which is tentatively assigned to the 14: 1 fatty alde- hyde; it represents a minor component of membrane phospho- lipids.

Quantitative evaluation of fatty acid composition was per- formed by half-height analysis of the peaks of gas chromatograms.

The carboxylic ester bond content was determined according to the method of Stern and Shapiro (24).

The fatty acid composition of phosphatidylcholine, phospha- tidylethanolamine, phosphatidglserine, phosphatidylinositides and sphingomyelin was determined after separation by thin layer chromatography as described above for total phospholipids.

For determination of sterols, Folch extracts of muscle homoge- nates and microsomes were fractionated by thin layer chroma- tography on Silica Gel G into phospholipids and neutral lipids using petroleum ether-ethyl ether-acetic acid (90 : 10 : 1, v/v) as solvent (25). The sterols were extracted from the plate with

chloroform-methanol (4 : 1, v/v) and evaporated to dryness (25). The residue was dissolved in chloroform and was analyzed on a model 402 Hewlett-Packard, F & i\l gas chromatograph using 3% QF, on Chromosorb Q (100 to 120 mesh) (26). Column temperature was 225”. The instrument was equipped with a flame ionization detector equilibrated to 245”. Helium was used as carrier gas at a flow rate of 60 ml per min. A Disc integrator was used for quantitative evaluation of the area under the peaks. Cholesterol represents the bulk of the sterols in muscle microsomes but the presence of smaller amounts of other sterols cannot be ruled out. Cholesterol and cholestanol had identical retention times in several systems tested.

RESULTS

Developmental Changes in Calcium Transport, ATPase Activity, and Phosphoprotein Concentration in Crude Microsome Prepara- tions Prepared by Diflerential Centrifugation-During 10 to 14 days of embryonic development the microsomes isolated from leg (Fig. I), superficial pectoralis (Fig. 2), heart (Fig. 3), and deep pectoralis (not shown) muscles of chicken embryos show little calcium transport or ATPase activity and the concentra- tion of phosphoprotein is barely measurable. In skeletal mus- cle microsomes (Figs. 1B and 2B), the ATPase activity meas- ured with RIg2+, lfgzf + Ca2+, or Ca2+ as activators rapidly in- creases during the 3rd week of incubation reaching maximum values of 3 to 4 pmoles per mg of protein per min at hatching, follwed by a gradual decline. The “estra” ATPase activity (Figs. 1A and 2A) calculated as the difference between the Mg* + Ca2+-activated and the magnesium-activated rate of ATP hydrolysis from the data of Figs. 1B and 2B increases with de- velopment at a slower rate than the total ATPase activity, maxi- mum values being reached only 2 to 3 weeks after hatching.

The rise in “e&a” ATPase activity in leg and superficial pectoral muscle microsomes during development was closely accompanied by an increase in the rate and extent of calcium accumulation (Figs. IA and 2A) and in the steady state con- centration of phosphoprotein intermediate measured with 5 RIM

Mgzf and 1OV M Ca2+ or with 5 mM Ca2+ as activators (Figs. 1C and 2C). Only marginal phosphoprotein concentrations were obtained with 5 mM MgClz and 0.5 mM EGTA as activators (Figs. 1C and 2C). The excellent correlation between extra ATPase activity, the rate of calcium transport and the steady state con- centration of phosphoprotein suggest that the concentration of calcium transport sites in the membrane increases with develop- ment. The data of Figs. 1 and 2 reveal a close similarity be- tween the calcium uptake capacity of the microsomes isolated from the white superficial pectoralis and the predominantly red leg muscles of chicken. This is unespected in view of the marked differences in Ca2+ transport between red and white mammalian muscle microsomes (2).

In agreement with the observations of Holland and Perry (la), the rise in magnesium + EGTA-activated (calcium-insensitive) ATPase activity of leg and superficial pectoral muscle micro- somes during the last week of incubation (Figs. 1B and 2B) occurs much earlier than the increase in the rate of calcium transport or in the level of phosphoprotein. Furthermore the calcium-in- sensitive ATPase (or ATPases) is associated with membrane fractions which are readily separated from the calcium-transport- ing elements of sarcoplasmic reticulum.

The maximum rates of extra ATPase activity and calcium transport of heart muscle microsomes (Fig. 3A) are much lower than those of their skeletal counterparts and show only minor changes with development. In keeping with the slow rate of

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1.5

1.2

0.9

0.6

0.3

DAYS OF DEVELOPMENT

1.0 0.8

0.6

0.4

0.2

2.0

1. 6

1.2

0.8

0.4

DAYS OF DEVELOPMENT

FIG. 1 (Zefl). Calcium uptake, ATPase activity, and phospho- protein concentration of leg muscle microsomes during develop- ment. The calcium uptake (A), ATPase activity (B), and phos- phoprotein concentration (C) were measured as described under “Experimental Procedures.” The rate of extra ATPase was cal- culated as the difference between measurements made in Mediums A and B (Fig. 1R) and is presented in Fig. 1A for comparison with the rate of calcium transport. A, O-O, calcium uptake after 1 min incubation; O-O, calcium uptake after 10 min incubation; A- - -A, extra ATPase; B, A---A, ATPase activity in Medium A; II---0, ATPase activity in Medium B; m---m, ATPase

calcium transport the maximum steady state concentration of phosphoprotein was 6 to 10 times lower in cardiac (Fig. 3C) than in skeletal muscle preparations. The prominent peak of mag- nesium-activated ATPase observed with skeletal muscle micro- somes at the time of hatching (Figs. 1B and 2B) is not apparent in cardiac systems.

The absence of major changes in the calcium transport func- tion of heart sarcoplasmic reticulum between 10 and 120 days of development is not surprising since the embryonic heart is fully functional already after 3 to 4 days of incubation. The marked changes in the calcium transport, extra ATPase, and phospho- protein concentration of skeletal muscle microsomes during the 1st week after hatching coincide with a sudden increase in mus- cular activity.

Sucrose Density Gradient Fractionation oj Leg Muscle Micro-

somes Isolated by DijJerential Centrifugation at Various Stages of Development-The crude microsome fraction obtained by dif- ferential centrifugation almost certainly contains membraneous elements other than sarcoplasmic reticulum. These may be derived from the surface membranes, T system tubules, or the mitochondrial outer membranes, and their relative amount is likely to vary during different stages of development.

Isopycnic sucrose gradient centrifugation of crude microsome preparations produces a continuous spectrum of vesicles ranging in average density from 1.05 to 1.20 (Fig. 4). The average density of microsome populations noticeably increases with de- velopment due presumably to an observed decrease in phospho- lipid to protein ratio (see below). Even after 120 days of development the chicken leg muscle microsomes occupy a rather broad region of density in comparison with adult rabbit skeletal muscle preparations isolated under identical conditions.

The fractions obtained by sucrose density gradient centrifu- gation display considerable differences in calcium uptake, ATPase activity, and the maximum concentration of phosphoprotein in- termediate (Table I).

-10 0 M 10 30 40 DAYS OF DEVELOPMENT

activity in Medium C; C, Q---Q, phosphoprotein concentration in Medium I; e--e, phosphoprotein concentration in Medium II; X--X, phosphoprotein concentration in Medium III.

FIG. 2 (center). The enzymatic activity of microsomes isolated from superficial pectoralis muscles at various stages of develop- ment. For details, see “Experimental Procedures” and the legend to Fig. 1.

FIG. 3 (right). The calcium transport, ATPase activity, and phosphoprotein concentration of heart microsomes isolated at various stages of development. For details, see “Experimental Procedures” and the legend to Fig. 1.

FIG. 4. Sucrose density gradient patterns of leg muscle micro- somes isolated at various stages of development. 1, 14-day-old embryo; B, 19-day-old embryo; S, la-day-old chick; 4, 16-day-old chick; 6, 120-day-old chick; 6, adult rabbit skeletal muscle micro- somes.

The specific activity of magnesium + EGTA-activated (Ca2+- insensitive) ATPase increases with decreasing density reaching a maximum in Fraction 5. This is a consistent observation throughout the whole range of development although the maxi- mum specific activity is found in microsomes isolated from 19- day-old embryos and 16-day-old chicks. On the other hand the Ca*+-sensitive “extra” ATPase which represents an increas- ing fraction of the total ATPase activity with development, ap- pears to be concentrated in the heavier gradient layers, and its contribution to the total ATPase activity decreases with de- creasing density. For example in microsomes from 120.day-old chicks 82.05% of the ATPase is Ca2+-sensitive in Fraction 1, 63.12% in Fraction 3, but only 2.430/, in Fraction 5. Unfortu- nately the extra ATPase activity of Fraction 1 represents only

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TAISLI': I Properlieaof microsomal subfractions obtained by sucrose density gradient cenlrijugation

of leg muscle microsomes at different slages of development

Microsomes obtained by differential centrifugstion were sep- which is frequently referred to as “sucrose gradient-purified mi- arated on sucrose gradients ranging from 0.3 to 1.5 M sucrose COW crosomes.” Extra ATPase is defined as the calcium-sensitive centration as described under “Experimental Procedures.” Frac- portion of Mg2+ + Ca2+-activated ATPase activity. Further de- tions 1 to 5 denote vesicles of decreasing density. Most of the tails on assay techniques may be found under “Experimental calcium-sensitive or extra ATPase is recovered in Fraction 3 Procedures.”

d+ uptake -r :a'+ uptake rate ,moles/ "F, protl "iIT

ca++ capacity pmales/mg prot/10 min

Phospha- protein inter- mediate mopes 32~1 1O'g protein

14 1 day 2 old 3 em- 4 brya 5

0.00-j 0. oo,, 0.006 0.006 0.003

0.039 0.065 0.0:7 0.OG.r 0.043

0.1qi; 0.114 0.191

19 1 3.2:; 0.021 0.037 0.135 day 2 7.19 0.030 0.195 0.390 old 3 39.75 0.02C 0.189 0.240 em- 4 12.72 0.017 0.071 0.145 bryo 5 3.62 0.027 0.032 0.200

16 1 1.83 0.016 0.028 day 2 7.99 0.048 0.046 old 3 42.iO 0.160 1.250 chicks 4 13.50 0.130 1.290

5 4.41 0.045 0.420

0.460 o.i;30 0.620 0.520

120 1 3.46 0.070 0.070 day 2 8.50 0.072 0.103 old 3 13.71 0.217 0.590 chicks 4 46.00 0.511 2.171

5 13.46 0.194 1.615

0.956 1.825 1.835 1.365

i-

ATPase mg protein

Mg*+EGTA activated

l- in

Extra

ATPaSe as per- cent of Mg+Ca activated ATPa*e

Mp+c a* activated .4wase, % of total

cat+ sensitive ATPase, % of total

0.020 0.260 0.377 0.507 0.466

0.250 0.010 0.381 -0.004 0.575 -0.068 0.513 -0.047

0.30 9.70

54.49 25.86

9.65

- -

-

- - 0.975 0.626 1.814 1.571 5.100 4.6GO 5.384 4.902

- 0.349 0.243 0.440 0.482

35.79 24.30

8.62 a.95

- - 4.63 13.78

43.95 48.98 39.54 28.38 11.88 8.85

0.350 0.303 0.047 13.42 0.43 0.49 0.692 0.445 0.247 35.69 3.73 11.19 1.515 1.157 0.358 23.63 43.54 86.49 3.890 3.866 0.024 0.61 35.38 1.83 5.678 6.033 -0.355 - lG.90 -

0.078 0.014 0.064 82.05 0.21 0.60 0.510 0.200 0.310 Go.78 3.35 7.18 1.410 0.520 0.890 63.12 14.94 33.26 1.450 1.000 0.450 31.03 51.53 56.40 2.880 2.RlO 0.070 2.43 29.97 2.57

0.6% of the total extra Al’l’ase and 65% or more of the total and calcium-sensitive Al’l’ase activities are recovered in Frac- tions 3 and 4 which contain most of the membrane mass. There- fore the value of this method as a standard purification procedure is quite limited.

Microsomcs purified on sucrose density gradient show similar dcvclopmental changes in calcium uptake, phosphoprot,eiu cotl- centration, and ATl’ase activity as the microsomes prepared by simple differential centrifugation (Figs. 1 to 3).

The rate of calcium uptake as well as the calcium capacity of microsomcs sharply increase after hatching together with the maximum steady state concentration of phosphoprotein and the extra ATl’ase activity. During the same time the phospholipid to protein ratio decreases.

The electron microscope appearance of microsomes prepared by differential celltrifugatiou (Fig. 5) is somewhat more hetero- geneous at all stages of development than the corresponding su- crose gradient fractions (Fig. 6), in generltl agreement with the biochemical findings. Microsomes isolated from leg muscles of chicken embryos contain numerous dense osmiophilic bodies (Fig. 5, A and B) which are absent after sucrose gradient ccw trifugation (Fig. GA).

Purification of Sarcoplasmic Reticulum dlembranes by Loading with Calcium Ozalale-Microsome preparations purified by dif- ferential and sucrose density gradient ceritrifugation were irl- cubated with calcium under conditions which permitted the active accumulation of large amounts of calcium oxalatc at the expense of ATl’ hydrolysis. The calcium oxalate containing vesicles were subsequently separated frotn the membrane ele- ments which are not capable of calcium accumulation by ceri- trifugation on a discontinuous sucrose gradient of 28 to 40% sucrose concentration. In this gradient the calcium oxalatc- containing vesicles form a pellet while the lighter calcium-free membranes stay in the supernatant. It is reasonable to assume that the calcium oxalate-loaded vesicles represent calcium-

transporting sarcoplasmic reticulum membranes in essentially pure form. Using this criterion, the calcium-transporting sarcoplasmic reticulum membrane mass expressed as percentage of the total mass of microsomal membranes increases with de- velopment from 2.6y0 in 14.day-old embryos to 17.2% just be- fore hatching, and stays close to 2Oci: up to 4 months of age (Table II). The amount of calciunl oxalate accumulated in the pellet fraction is 20 to 30 times greater than that found in the microsomal vesicles of the supernatatit and increases from 1.70 pmoles per mg of protein it1 19-day-old embryos to 6.92 kmoles per mg of proteiti iii microsomcs isolatctl from the leg muscles of a 4-month-old chicken. Repeated tests of the cal- cium uptake activity of the supernatant fract,ion after separation yielded data similar to those prcsentcd in Table II. These data together with those obtained 011 crude leg muscle microsomes (Fig. 1A) suggest that cvcn in muscles of adult animals less than one-third of the mcmbranc mass of microsome preparations isolated by simple diffcretitial cctitrifugation represent calcium- accumulating structures. Ilowcver this may be a minimum estimate. The difference bctwcen crude microsomes and the calcium osalate pellet fraction is less pronounced with respect to the extra Al’l’asc activity (Table I1 I). The supcrnatant frac- tion obtainctl after the removal of calcium osalate-loaded vesi- cles still retailis sizable calcium-sensitive “cxtm” ATl’ase ac- tivity (Table III). This observation may imply the presence of microsomes in the supcrnatatlt which contain calcium pump but are unable to accumulate (:a*+, perhaps due to the leakiness of the membrane.

The customary increase in calcium uptake and extra Al’l’ase activity with development is accompanied by a comparable in- crease in the concentration of phosphoprotein (Table II) which reaches considerably higher steady state concentration in 4- month-old chicken leg microsomes purified by calcium oxalate sedimentation than in either crude or sucrose gradient-fraction- ated preparations.

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FIG. 5. Electron micrographs of microsomes prepared by dif- ferential centrifugation from leg muscles of A, IO-day embryos; B, B-day embryos; C, B-week chicks. The majority of the vesicles have diameters of 1,000 to 2,000 A. The microsomes show less variability in size and density with development. Particularly noticeable is the preponderance of sharply defined, elongated membrane profiles and the absence of dense osmiophilic bodies after hatching. Magnification is approximately X 32,000.

TABLE II

Phosphoprotein concentration and Ca2+ transport in microsomal

subfractions isolated after calcium oxalate loading

The fractionation and the various assays were performed as described under “Experimental Procedures.”

Stage of development

14-day embryo. . 19-day embryo. ZO-day embryo. . 16-day chick.. 120-day chick..

-

.-

-

Protein in calcium oxalatc sediment BS per

cent of total protein

2.60 7.93

17.20 16.43 19.58

-

s1

Cal+ uptake

1.70

0.195 4.01 0.215 6.92

- P

_-

C

.- 1

hosphoprotein Protein Composition of Muscle Jlicrosomes during Develop- intermediate ment-Polvacrvlamide gel electronhoresis of microsomes isolated

from leg &IS&S by diffkrential c&trifugation, showed a marked Mcium oxalate

sediment increase in the concentration of the M protein with development (Fig. 7). The M protein was previously identified in rabbit

moles “=P/loe g skeletal muscle microsomes with the ATl’ase enzyme involved pro1ein in the calcium transport (27). The change in the concentration

0.68 of M protein closely accompanied the increase in extra ATl’ase activity, calcium transport, and the concentration of phospho-

FIG. 6. Microsomes prepared by sucrose density gradient (Frac- tion 3) from leg muscles of A, 14-day embryos; 11, 19-day embryos; C, 6-week chicks. These preparations appear more homogeneous, especially at early stages of development (Fig. 6A ) than micro- somes isolated by differential centrifugation (Fig. 5A). Note the absence of osmiophilic bodies in Fig. 6A. Magnification is ap- proximately X 32,000.

The electron microscope appearance of the calcium-accumu- lating membrane material in thin sections (not shown) is not readily distinguishable from sucrose gradient-fractionated micro- somes (Fig. 6), except that early embryonic preparations ob- tained by sucrose gradient fractionation are slightly more hetero- geneous.

1.29 3.03

protein. During the same period there was some decrease in the relative amount of small molecular weight proteins which do not have clearly assigned functional significance. Essentially

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618 TABLE III

ATPase activity of microsomal subfractions isolated after calcium oxalate loading

For details see “Experimental Procedures.”

Mg* + C&-activated ATPase Mgz+ + EGTA-activated ATPase Extra ATPase

Stage of development Calcium oxalate Calcium oxalate Calcium oxalate Calcium oxalate Calcium oxalate Calcium oxalatc

supernatant sediment supernatant sediment supernatant sediment

p??WleS Pi/V&g firOtein/Vti?S pmoles Pllmg ~rotcin/min pm& P,/mg fwoteinjmin

14-day embryo. 0.265 0.260 0.220 0.260 0.045 0 19-day embryo. 1.30 0.73 1.44 0.460 0 0.27 16-day chick.. 1.64 0.71 1.30 0.26 0.34 0.45 120-day chick.. 0.95 0.89 0.42 0 0.53 0.89

4

Days of Development

M-

14 19 26 33 42 65

Days of Development

FIG. 7. Protein composition of muscle microsomes during de- velopment. Polyacrylamide gel electrophoresis was carried out as described under “Experimental Procedures.” A, leg muscle microsomes; B, heart muscle microsomes. M indicates the posi- tion of transport ATPase.

identical developmental changes were observed in the protein composition of microsomes isolated from superficial pectoralis and deep pectoralis muscles.

In heart muscle microsomes the developmental changes in pro-

Extra ATPase as per cent of Mg*’ + &*+-activated ATPase

Calcium oxalate Cal$;gmynlate supernatant

16.98 0 0 36.98

20.73 63.38 55.78 100.00

tein composition were much less pronounced in accord with the nearly constant ATPase activity, calcium transport, and phos- phoprotein concentration (Fig. 7).

As purification of microsomes by sucrose gradient centrifuga- tion or calcium oxalate sedimentation permits the separation of fractions with varying calcium transport and ATPase activity, the protein composition of the different membrane fractions iso- lated from leg muscles during development was investigated. Surprisingly only minor differences are apparent between the protein composition of crude microsomes subjected only to dif- ferential centrifugation (Fig. 7), microsomes purified by sucrose gradient centrifugation (Fig. 8A), the calcium oxalate pellet which represents pure sarcoplasmic reticulum membranes (Fig. 8B), and the calcium oxalate supernatant (not shown) which contains particles that are essentially devoid of calcium transport activity.

The developmental changes in the protein composition of crude microsomes (Fig. 7) are also reflected in the protein com- position of sucrose gradient-purified (Fig. 8A) or calcium oxa- late-sedimented preparations (Fig. 8B). Even sucrose gradient fractions of widely differing densities which showed large dif- ferences in the specific activity of calcium-sensitive ATPase and in phosphatidylcholine and sphingomyelin content had similar protein compositions (Fig. SC).

Phospholipid Content of Microsomes at Various Stages of Development-During the early embryonic period the microsomal membranes isolated from leg muscles contain relatively large amounts of phospholipids (Fig. 9). The phospholipid to pro- tein ratio declines during development to 0.6 to 0.7 pmole of lipid-P per mg of protein and remains at that level from 2 weeks to 4 months after hatching. There is only slight difference in the phospholipid to protein ratio of crude microsomes obtained by differential centrifugation and subfractions isolated by su- crose gradient or by calcium osalate loading. The major de- crease in phospholipid to protein ratio coincides with the sharp increase in ATPase activity and calcium transport around the time of hatching. Part of this change is attributable to the in- creased concentration of the YI protein in the membrane. The decrease in phospholipid to protein ratio during development is reflected in the increased density of microsomes as determined by sucrose density gradient centrifugation.

The carbosylic ester bond content of the membrane deter- mined on crude microsomes (Fig. 9) is about that expected from the phospholipid content at all stages of development. Con- sequently the di- and triglyceride content of the preparations is probably negligible. Esterified cholesterol was not detected.

The principal phospholipid fractions were identified by two- dimensional thin layer chromatography as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, sphingomyelin,

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I 2 3 4 ) 5 6 7 8 1 9 IO II 12

FIG. 8. Protein composition of microsomal subfractions at 14-day-old embryo; 6, 19-day-old embryo; 7, 12-day-old chick; 8, various stages of development. A, sucrose gradient Fraction 3. &week-old chick. C, sucrose gradient fractions of leg muscle I, 14-day-old embryo; 2, Wday-old embryo; 5, 16-day-old chick; microsomes from 4-month-old chicken. 9, Fraction 1; 10, Frac- 4, 120-day-old chick: B, calcium oxalate sediment fraction. 6, tion 2; 11, Fraction 3; 12, Fraction 4.

Days of development

FIG. 9. The phospholipid to protein ratio of microsomal mem- I branes during development. The phospholipid content of micro- PC \

somes was measured after Folch extraction as described under 9 .PI

“Experimental Procedures.” For the measurement of carhoxylic “$M :

ester bonds, the method of Stern and Shapiro was used (24). PS O-O, crude microsomes; ~-m, peak fraction after sucrose : I density gradient centrifugation; A--A, sediment after calcium \

oxalate loading; A--A, supernatant after calcium oxalate load- h \5&

ing; +- - -+, carboxylic ester bonds. FIG. 10. Thin layer chromatogram of microsomal lipids isolated from sucrose gradient-fractionated leg muscle microsomes of

and phosphatidylinositides (Fig. 10). The relative amounts of 14-day-old embryos (A) and 5-week-old chicks (B). PC, phos-

these phospholipids shows only minor changes with development. phatidylcholine; PE, phosphatidylethanolamine; PS, phospha- tidylserine, SM, sphingomyelin; PI, phosphatidylinositol; NF,

A quantitative comparison of the phospholipid content of neutral fat.

crude microsomes and their subfractions obtained by sucrose density gradient centrifugation or calcium loading at various stitutes 2 to 10% of the total phospholipids showed slight de- stages of development is shown in Fig. 11. In general, there is crease during development. less phosphatidylcholine and more phosphatidylethanolamine Although there were no striking differences in phospholipid and phosphatidylserine in chicken than in adult rat or rabbit composition between crude microsomes and the purified frac- skeletal muscle microsomes. Phosphatidylcholine comprises tions represented in Fig. 11, a systematic analysis of all sucrose about 40 to 50~~ of microsomal phospholipids and remains es- gradient fractions revealed considerable heterogeneity. Sucrose sentially constant during development. Further work is re- gradient fractions obtained from leg muscle microsomes of 19- quired to evaluate the significance of the slight developmental day-old embryos and 4-month-old chickens show increasing changes observed in the concentration of phosphatidylethanol- phospholipid to protein ratios with decreasing density (Table amine (25 to 30%). The combined phosphatidylinositide and IV). Fraction 3 contains usually most of the calcium transport sphingomyelin fraction and phosphatidylserine which con- and extra ATPase activity. The contribution of calcium-

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620

I I I , I , I I I I I I I

Phosphatidylchaline Phosphatidylserine

z .A? lo- m 5 z -z

phosphatidylethanolamine sphingomyelin + inositides

; 50- a

An-

Days of development

FIG. 11. Phospholipid composition of microsome preparations at various stages of development. For technical details, see “EX- perimental Procedures.” O-0, crude microsomes; O--O, peak fraction after sucrose density gradient centrifugation; O--O, sediment after calcium oxalate loading; +m, super- natant after calcium oxalate loading. Unidentified phospholipids accounted for less than 127, of the total lipid-P.

TABLE IV Phospholipid composition of membrane subfractions obtained by

SUCTOS~ gradient centrifugation

Sucrose density gradient centrifugation was performed as de- scribed under “Experimental Procedures.” Fractions 1 to 5 represent progressively lighter portions of the gradient.

Age

19.day embryo

120-day chicken

1 2 3 4 5

1 2 3 4 5

Phos- holipic

to xotein ratio

Sphing- Phos- omye- pha- tidyl-

holine etha.?ol- serine amme tidyl- inositol

-l-l-I---

0.50 0.83 1.20 2.30

45.67 22.18 7.08 9.48 15.56 46.68 24.58 8.24 13.56 6.92 37.33 24.77 10.03 19.25 8.78

0.28 55.60 6.50 10.06 7.55 22.25 0.45 60.00 24.30 7.30 1.85 5.65 0.63 55.50 20.70 8.80 5.50 9.50 0.86 47.60 25.50 7.90 13.20 7.70

Phospholipid content, per cent of total lipid-P

sensitive ATPase to the total ATPase activity decreases with decreasing density (Table I) reaching a minimum value in Frac- tion 5 which also has the highest sphingomyelin and lowest phosphatidylcholine content. Since more than 75% of the total microsomal mass is represented by Fraction 3 together with most of the calcium transport and ATPase activity, the fractions with the most unusual phospholipid to protein ratios do not have great influence on the average values reported in Fig. 11.

16:0

I B 16:O

I

IS:0 I

l&2 ’ IS:1 ,

F

7 20:4 16.1

L \

FIG. 12. Gas chromatogram of methylated fatty acids isolated from total lipids of sucrose gradient-fractionated leg muscle mi- crosomes of 14-day-old embryos (A) and 16-day-old chicks (B). The peak marked ? is unidentified but on the basis of its mass spectrum probably represents the dimethyl acetal derived from the 14:l fatty aldehyde. All other components were identified by their retention time and mass spectra.

Fatty Acid Composition of Microsomal Phospholipids during

Development-In view of the well known influence of fatty acid chain length and unsaturation upon the permeability of artificial lipid membranes for Ca zf (28) developmental changes in the fatty acid composition of sarcoplasmic reticulum membrane phospholipids were investigated by gas-liquid chromatography. Typical examples of the separation of fatty acid methyl esters are shown in Fig. 12 representing sucrose gradient-purified microsomes from leg muscles of 14-day-old embryos and 4-month- old chickens, respectively. There is a striking decrease in palmi- tate (16 :0) and increase in linoleate (18 :2) content with develop- ment. The data are summarized in Fig. 13A for crude and in Fig. 13B for sucrose gradient-fractionated microsomal lipids over a period of development ranging from lo-day-old embryos to 4-month-old chickens. In microsomes isolated from leg muscles of lo- to 11-day-old embryos the saturated fatty acids (palmitate and stearate) constitute about 54% of the total esterified fatty acids. During the next 2 to 3 weeks of develop- ment the concentration of palmitate decreases while that of linoleate increases so that in the microsomal lipids of 12.day-old chicks the saturated fatty acids represent only 37% of the fatty acid content. The short chain unsaturated fatty acids are minor components representing less than 5% of the total fatty acids. The major unsaturated fatty acids in addition to lin- oleate are oleate (18:l) and arachidonate (20:4) which show some decrease with development. In chicken microsomes fatty aldehydes are not found in amounts reported for rabbit sar- coplasmic reticulum (29) and on the basis of mass spectra only the 14 : 1 aldehyde appears to be present in significant quantity.

The net result of the developmental changes in fatty acid composition is that the early embryonic membranes containing large amounts of saturated fatty acids are converted by the 30

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to 35 days of development into calcium-transporting structures in which about 60% of the fatty acids are unsaturated. This cannot be attributed to dietary factors alone since the changes in the palmitate and linoleate content are already evident be- tween 14 and 20 days of development, i.e. before hatching.

Fatty Acid Composition of Phosphatidylcholine and Phos-

phatidylethanolamine in Various Microsomal Fractions during

1

DAYS OF DEVELOPMENT

FIG. 13. Fatty acid composition of total lipids in crude (A) and sucrose density gradient-fractionated (B) leg muscle microsomes as function of development. The fatty acid composition of ex- tracts prepared according to the method of Folch was analyzed by gas-liquid chromatography after methanolysis according to the method of Keenan and Morre (23). 0, palmitic; n , stearic; 0, oleic; 0, linoleic; A, arachidonic; f, unidentified; Q, palmitoleic. The unidentified fraction has a retention time similar to that of methyl myristoleate but on the basis of its mass spectrum it is tentatively identified as the dimethyl acetal derived from the 14:l fatty aldehyde.

621

Development--The fatty acid composition of the two major microsomal phospholipids shows characteristic differences (Table V). Phosphatidylcholine contains more palmitate but less stearate and arachidonate than phosphatidylethanolamine. At 14 days of embryonic development, saturated fatty acids (palmi- tate and stearate) constitute about 54 and 35% of the total fatty acids in phosphatidylcholine and phosphatidylethanol- amine, respectively. The corresponding values at 33 days of development are 46 and 33%, respectively. l’hosphatidyl- ethanolamine contains more unsaturated fatty acids, primarily arachidonate, at an early embryonic stage than lecithin and changes relatively little during subsequent development. The concentration of linoleate and the increase in linoleate content during development are of similar magnitude in the two classes of phospholipids. The fatty acid composition of phospholipids isolated from crude microsomes or from purified fractions ob- tained by sucrose density gradient centrifugation or calcium oxalate loading are practically identical. Insufficient amounts of material permitted only semiquantitative analysis of the fatty acid composition of phosphatidylserine, which resembles that of phosphatidylethanolamine. Phosphatidylinositol and sphingomyelin analyzed as combined fractions had a fatty acid composition similar to lecithin.

Sterol Content of Leg iWuscle illicrosomes during Development-

The sterol contents of whole muscle homogenate, crude mi- crosomes, and the various microsome subfractions were analyzed at four stages of development after thin layer chromatographic separation by gas-liquid chromatography. No esterified sterols were detected and a major part of the sterol content corresponds to cholesterol. In whole muscle homogenates the sterol content decreases with development from 0.261 mg per g of muscle in 14-day-old embryos to 0.172 mg per g of muscle in 35.day-old chicks (Table Vl). It is generally assumed that a major part of the cholesterol is associated with the cell surface membrane and therefore the observed decrease in cholesterol content may result from the increase in the size of muscle cells during develop- ment.

TABLE V Fatty acid composition of phosphatidylcholine and phosphatidylethanolamine in chicken skeletal

muscle microsomes isolated at various stages of development

Phospholipid extracts prepared according to Folch et al. (19) were fractionated by thin layer chromatography. The separated

according to the method of Ackman (21). Phosphatidylserine, phosphatidylinositide, and sphingomyelin together with small

phospholipids were subjected to methanolvsis and the methvl es- ters of fatty acids were separated by gasliquid chromatography

amounts of unidentified uhosnholioids accounted us11a.11~ for less than 207; of the phosphohpidcontent at all stages of development.

Stage of Development

14 day chick embryo

18-19 day chick embrya

1 day chick

6 day chick

12 day chick

16 day chick

18 day chick

35 day chick

42 day chick

120 day chick

Microsome Fraction

Crude Sucrose gradient ca-oxa1at.2

loaded

Crude Sucrose gradient Ca-oxalate loade<

Crude

Crude

Crude Ca-oxalate loadec

Sucrose gradient

Crude

Ca-oxalate loaded

Crude

Sucrose gradient

Phosphatidylcholine

14:o 14: l(?) 16:O 1G:l 18:O 18:l 18:2 20:4

0.82 0.59 43.91 3.70 9.69 19.96 11.24 10.09 0.74 Tr. 47.11 2.17 9.45 20.10 9.44 10.98 0.30 TI. 50.93 0.84 9.78 18.99 9.72 9.49

1.72 2.84 30.83 3.04 12.84 17.07 18.75 12.92 1.06 1.77 35.29 1.88 12.20 19.64 15.44 12.72 Tr. T IT . 32.25 1.73 14.33 19.98 20.32 11.40

1.80 2.20 28.62 3.20 13.79 20.83 18.99 10.57

0.92 0.88 27.77 2.56 15.32 17.92 25.78 8.84

0.28 Tr. 29.08 2.26 17.13 18.16 Tr. Tr. 21.18 2.27 17.86 22.18

0.70 0.88 32.34 2.45 13.43 19.27 27.44 3.51

Tr. Tr. 29.45 2.13 15.75 20.66 28.95 3.OG

0.47 0.68 25.24 2.43 15.88 18.88 31.11 5.34

0.56 1.29 33.49 1.22 13.83 16.34 26.48 6.80

0.30 3.01 30.89 1.55 11.11 21.85 19.24 12.01

Phosphatidylet~anolamine

14:o 14:1(?) 16:o 16:1 18:0 18:l 18:2 20:4

1.39 7.58 13.96 2.64 21.37 14.57 10.83 27.67 1.28 5.40 17.67 2.19 24.G 12.68 5.53 30.59 2.03 Tr. 17.55 Tr. 27.51 13.69 11.22 28.03

:::: 6.78 8.13 15.37 16.47 3.13 3.79 20.84 18.15 13.20 12.84 19.05 13.33 21.54 22. VG 1.20 1.82 15.59 2.09 20.29 13.00 23.55 22.17

2.54 5.42 16.49 3.86 16.92 16.40 19.84 18.57

1.44 4.11 11.46 1.71 20.67 11.53 23.21 25.89

1.37 2.34 12.47 2.18 20.91 12.64 28.41 19.69 1.60 0.34 11.52 2.27 25.30 11.29 27.15 20.53

1.29 6.32 10.31 1.50 22.61 11.39 18.93 27.65

1.33 3.59 11.74 2.07 23.23 11.66 27.25 19.15

0.73 2.25 9.15 0.97 23.03 11.46 29.60 22.82

1.66 5.33 10.59 1.29 21.57 9.41 27.81 22.35

1.86 7.28 11.05 3.21 17.50 14.88 16.57 27.69

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622

TABLE VI

Sterol content of leg muscle microsomes during development

Folch extracts were fractionated by thin layer chromatography into classes of phospholipids and neutral lipids. The sterols were extracted with chloroform-methanol (4:1, v/v) according to the method of Skipsky et al. (25) and subjected to gas-liquid chromatog- raphy as described by Elliott et al. (26).

Stage of development Muscle homogenate Crude microsomes Peak fraction from sucrose Calcium-oxalate-loaded Supernatant from calcium density gradient microsomes oxalate-loading

experiment I I I

mg choleslerol/g llrd wt muscle mg cholesterollmg firolcin or (wdes phospholipid/pmole cholesterol)

14-day embryos. 0.261 0.144 (1.99) 0.155 (1.95) 0.129 (3.31) 0.206 (1.86) 1% to 19-day embryos.. 0.233 0.119 (2.80) 0.087 (3.60) 0.059 (4.54) 0.099 (2.85) 12-day chicks.. . . . 0.197 0.068 (4.15) 0.041 (5.85) 0.029 (7.73) 0.035 (6.44) 35-day chicks. . . . 0.172 0.065 (4.22) 0.049 (5.35) 0.031 (7.50) 0.044 (5.88)

Crude microsomes and the peak fraction obtained by sucrose gradient centrifugation contained significantly larger amounts of cholesterol than sarcoplasmic reticulum membranes prepared by calcium oxalate loading. This difference is equally evident from the cholesterol to protein or from the phospholipid to cholesterol ratios.

The data suggest that sarcoplasmic reticulum membranes contain a small but significant amount of cholesterol. There is a sharp decrease in the cholesterol content of all microsome prep- arations during development which coincides with the increase in calcium transport and extra ATPase activity and with the changes in the phospholipid to protein ratio of the membrane. No significant further change in cholesterol was observed be- tween 33 and 56 days of development.

DISCUSSION

Developmental changes in the protein composition, ATPase, and calcium transport activity of muscle microsomes provide a slow motion picture of the assembly of functionally competent sarcoplasmic reticulum membranes endowed with calcium transport activity.

At 10 to 14 days of embryonic development the ATPase and calcium transport activity of microsomes isolated from leg, superficial pectoralis, and deep pectoralis muscles are low, com- parable to that of the heart. The membranes contain primarily low molecular weight proteins and on gel electrophoresis the M band which is known to contain the calcium transport ATPase is only a minor protein component. These embryonic mem- branes are rather rich in phospholipids and cholesterol and the fatty acid content of phospholipids is characterized by relatively high concentration of palmitic and stearic acid.

In skeletal muscle microsomes between I4 and 30 days of development there is a spectacular increase in the extra ATPase activity, phosphoprotein concentration, and Ca2+ transport together with an increase in the concentration of hI protein, which in l- to 2-week-old chicks constitutes already 60 to 70% of the protein content of microsomes. Parallel with these changes the phospholipid and cholesterol content of the mem- branes decreases and there is a large increase in the linoleate content of membrane phospholipids with corresponding decrease in palmitate. The concentration of arachidonic acid decreases to a lesser extent. These changes in the unsaturation and average chain length of the fatty acids of microsomal phos- pholipids may be of importance in maintaining the proper flu- idity of the lipid phase of microsomal membranes at physiological temperatures.

In heart muscle microsomes the developmental changes in

protein composition and enzymatic functions are much less pronounced.

The average concentration of the various phospholipid classes remains relatively constant during the rather striking changes in the fatty acid composition of the membrane. Phosphatidyl- choline is characterized by higher palmitate and lower stearate and arachidonic acid content than phosphatidylethanolamine. The developmental changes in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine are qual- itatively similar. It is unlikely that the observed alteration in fatty acid composition during development could be explained by dietary factors alone since significant changes are apparent before hatching. It is possible however that the unusual aspects of the fatty acid composition of 120.day-old chick microsomes could have resulted from dietary factors.

Phosphatidylserine had fatty acid composition similar to phosphatidylethanolamine. This is not surprising in view of the common biosynthetic pathway shared by these two phospho- lipids. The phospholipid composition of the combined sphingo- myelin-phosphatidylinositol fraction was qualitatively similar to that of phosphatidylcholine.

The developmental changes in fatty acid composition are of similar magnitude in crude, or sucrose density gradient- and calcium oxalate-fractionated microsomes indicating that all membrane systems which are represented in crude microsome preparations participate in the changes to a similar extent.

Sarcoplasmic reticulum membranes defined as the rapidly sedimenting material obtained after calcium oxalate loading account for an increasing fraction of the total microsomal mem- brane mass with development. This may contribute to the striking increase in the homogeneity of vesicle populations ob- served by electron microscopy in microsome preparations iso- lated from older animals.

The increase in calcium transport and calcium-sensitive ATP- ase activity is preceded during development by the appearance of a magnesium-activated ATPase enzyme which does not require Ca2+ for activity and reaches its maximum rate shortly before hatching. The subsequent decline in the activity of this enzyme during development correlates well with the enhance- ment of the Cazf transport-related functions and with the in- crease in the average density of microsomal membranes. The calcium-insensitive ATPase is obtained with the highest specific activity in the lighter sucrose gradient fractions, while the cal- cium-sensitive transport ATPase is localized primarily in the heavier layers.

Several interpretations of these observations are possible. 1. The calcium-insensitive ATPase may be entirely independ-

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ent from the calcium pump and performs some metabolic func- tion associated with development.

2. The calcium-insensitive ATPase may be a precursor of the transport ATPase which acquires calcium sensitivity at a rel- atively late stage of development. The hypothetical “coupling factor” which links ATP hydrolysis to Ca2+ transport and im- parts Ca2+ sensitivity upon the system may be a protein subunit which links the various elements of the Ca2+ pump into a func- tioning complex. The accumulation of M protein of 100,000 molecular weight with the appearance of Ca*+-sensitive ATPase and calcium transport activity during later development may represent the assembly of fully developed Ca*+-sensitive ATPase enzyme from preformed subunits. Alternatively the assembly of the transport ATPase into a functional enzyme complex may proceed only when the phospholipid and fatty acid composition of the membrane becomes optimal.

The apparent correlation between the appearance of ATPase enzyme molecule and the change in the fatty acid composition of phospholipids with development is consistent with most con- ceivable mechanisms of membrane assembly. The following possibilities may be considered.

1. The simplest assumption is that the synthesis of lipid and protein constituents of the membrane occurs independently but is so coordinated that the fatty acid composition of the mem- brane becomes optimal when the transport ATPase is fully developed.

2. Alternatively, although the synthesis of ATPase enzyme protein or its subunits is independent of the fatty acid com- position of membrane phospholipids the coupling of ATl’ase activity to Ca*+ transport, the assembly of the enzyme from its subunits or the insertion of the enzyme protein into the mem- brane may require some unsaturated fatty acids. The de- pendence of calcium-sensitive ATPase activity upon phos- pholipids is well known (30). The evidence concerning specific requirement for unsaturated fatty acids in the in vitro recon- stitution of calcium transport and ATPase activity from lipid- depleted membranes is confiicting (8, 9, 16, 31).

It seems unlikely that the phospholipid requirement for the appearance of calcium transport during development would be related to a simple permeability function of phospholipids since phospholipids containing saturated fatty acids are excellent permeability barriers for Ca *+ (28). The calcium permeability of microsomal membranes is four to six orders of magnitude greater than that of pure phospholipid bilayers. Therefore other membrane components, presumably proteins, are the principal determinants of ion permeability under physiological conditions.

Considering the evidence so far available the developmental changes in Ca*+ transport and ATPase activity are consistent with a stepwise assembly of the calcium transport system from preformed subunits which are present in the membrane before significant calcium transport becomes demonstrable. Although

the requirement for phospholipids in ATPase activity and cal- cium transport is well established, their possible effects upon the interaction between the subunits of the Cazf pump requires further investigation.

The presence of cholesterol in purified sarcoplasmic reticulum preparations suggests but does not fully establish that cholesterol is a genuine membrane constituent of sarcoplasmic reticulum. Contamination of the calcium oxalate sediment fraction with

623

less than 20% surface membrane could account for the observed cholesterol content. Since cholesterol is not required for ATP hydrolysis or calcium transport (32), the significance of the developmental changes in cholesterol content remains to be established.

Acknowledgments-Our thanks are due to Dr. W. H. Elliott for his help with the analysis of sterols and the use of mass spec- trometer, to Mr. C. E. Bussmann for the evaluation of mass spectrograms, and to Shirley Carroll for assistance with the electron microscopy.

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Ricardo Boland, Anthony Martonosi and Thomas W. TillackReticulum

Developmental Changes in the Composition and Function of Sarcoplasmic

1974, 249:612-623.J. Biol. Chem. 

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