Variation in the Lipid Composition of Rabbit Muscle Sarcoplasmic ...

Variation in the Lipid Composition of Rabbit Muscle Sarcoplasmic Reticulum Membranewith Muscle Type* (Received for publication, June28, 198'2)

Douglas Borchman, Robert Simon, and Ellen Bicknell-Brown From the Chemistry Department, Wayne State Ilniuersity. Detroit, Michigan 48202

The compositions of sarcoplasmic reticulum (SR) membranes from rabbit caudofemoralis, tibialus, and soleus muscles (fast, mixed, and slow twitch, respectively) were analyzed. Compared to caudofemoralis (fast twitch) SR, soleus (slow twitch) SR contained a significantly greater percentage of cholesterol, phosphatidylinositol, and sphingomyelin and a lesser percentage of phosphatidylcholine. Correlations between properties reported for the SR isolated from different muscle types and our analyses of the compositions are discussed. We suggest that the greater cholesterol content and the greater sphingomyelin to phosphatidylcholine ratio present in soleus SR contribute to decreased bilayer fluidity and, hence, decreased ea"+ATPase activity.

plasmic reticulum (E, washed) was prepared from isolated muscles (described below) of 11 5-8-pound male white New Zealand rabbits according to the procedure of MacLennan (14) with the following modifications suggested by Banerjee et al. (15). 5 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, pH 7.5, 0.12 M NaC1, 0.5 mM phenylmethanesulfonyl fluoride was used as extraction buffer and 5 mM 4-(2hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.5, 0.3 M sucrose was used as storage buffer.We found, as did Banerjee et a1. (15), this preparation can be stored for at least several months at -70 "C without loss of ATPase activity. However, all samples were analyzed within 1 week of the sarcoplasmic reticulum preparation. The protein concentration of the various suspensions, where required in MacLennan's procedures (14), was measured by Spector's refinement of the Coomassie blue method (16). Because the soleus and the tibialus muscle mass/rabbit is less than required for sufficientlv accurate measurements for of the all analyses undertaken, rabbits were usually pooled in groups of two for soleus and tibialus muscle. For caudofemoralis muscle and for combined muscle measurements, analysesof individual rabbits were performed. leg and back muscles The activity of SR' Ca2+-ATPaseand the efficiency of the The "combined" muscle group includes the hind the caudofemoralis, tibialus, and soleus. Muscle masses were SR Ca2+p u m p h a v ebeen linked to SR lipid composition both minus of MacLennan (14) measured tokO.1 g in accuracy and the procedure in a very specific way by head group requirement and in a was scaled proportionally for each muscle preparation. general way by bilayer fluidity requirement (1).Abrupt enProteinDetermination-Measurements of proteinaccording to hancement of the SR Ca"-ATPase activity has been observed Lowry et al. (17) were made in order to measure lipid/protein ratios at 15 t o 20 "C (2, 3), where the SR lipid bilayer is believed to for the various samples. Bovine serum albumin was used as standard. Lipid Extraction and Charactmiration-1 ml of H I washed (14) become entirely liquid crystalline.? sarcoplasmic reticulum suspension (0.5 to 15 mg of protein/ml) was The activity of Ca"-ATPase bound to SR from different added to 3 ml of CHCI:3/MeOH (12, v/v) and vortexed I min. T h e m u s c l e t y p e s is quite variant. The activity of ATP hydrolysis addition of 1 ml of CHCl, was followed bv I-min vortexing, then and the e n z y m e p h o s p h o r y l a t i o n and dephosphorylation kiaddition of 1 ml of H,O, then I-min vortexing. The phases were netics f o r Ca'+-ATPase isolated f r o m the caudofemoralis sar- separated by centrifugation. T h e lowerphasewasusedforlipid analyses. Care was taken not to disturb the white, denatured protein coplasmic reticulum are quite different from that of the Ca'+ATPase isolated f r o m the tibialus and soleus SR (4-7). His- interface. From the lipid extract, cholesterol was quantified colorimetrically tochemically, the caudofemoralis is f a s t t w i t c h type muscle; at 550 nm using the Liebermann-Burchard reaction (IS). the tibialus is a mixed fast twitch-slow twitchmuscle, and the Lipids were separated by two-solvent phase, unidimensional high soleus is a s l o w t w i t c h t y p e muscle (4).The present s t u d y performance thin layer chromatography on Scientific Products silica well defined bands for both investigates the possibility that the differences in Ca2+-ATP- gel G plates (19).These plates gave sharp, ase activity in the various types of SR may be due in part or lipid standards and extracts. Sample phosphate measurements were in whole to differing SR lipid composit,ions. We h a v e , there- corrected for silica gel background phosphate determined from blanks to at sites adjacent to sample bands. Corrections required amounted fore, isolated and analyzed the phospholipid and cholesterol less than 3 8 of the measured sample phosphate. The amount of for caudofemoralis, tibialis, and soleus muscle SR membranes phosphate for each lipid type and the total lipid phosphate in the in order to c o m p a r e their lipid compositions with their CaL"- lipid extract were determined by the method of Parker and Peterson ATPase activities. Because previous analysesof lipid compo- (20). Integrity of the R I WashedSarcoplasmicReticulumPreparcrsition were performed o nc o m b i n e d muscle t y p e ,w e also tion-Sodium dodecylsulfate-gelelectrophoresiswascarriedout m e a s u r e d lipid composition for c o m b i n e d muscle in order to according to the methodof Weber and Osborn (21). Sodium dodecyl c o m p a r e our results with previously published results (8-13). sulfate-gel electrophoretic profilesof the SI3 preparations were comparable to those obtained by MacLennan (14). One major 102-kiloEXPERIMENTALPROCEDURES dalton CaL+-ATPase band and two minor bands, assigned to calsequestrin and calcium-binding protein, were observed. As expected, Preparation of Sarcoplasmic Reticulum-Fragmented sarco(15), only the major Ca"upon octyl glucopyranoside purification * This work was supported by National Institutes of Health Grant ATPase band was observed. Infrared spectra of the octyl g1ucop.vranoside-purified Ca"-ATPase showed characteristic lipid and proGM 27243. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be tein peaks. Ca"-ATPase activity was measured a t 37 "C in the reactiun mehereby marked "advertisement" in accordance with 18 U.S.C. Section dium used by Hidalgo et al. (3). Liberated phosphate was determined 1734 solely to indicate this fact. ' The abbreviations used are: SR, sarcoplasmic reticulum; Ca"- using the method of Fiske and SubbaItow (22). The activity of our preparations at 37 "C in Hidalgo's medium (3) ATPase, Ca"-dependent ATPase;PC, phosphatidylcholine; PI, phosaveraged 3.8 IU/mg 0.8 S.D., comparing favorably with the 2.9 IU/ phatidylinositol; SM, sphingomyelin. mg of ATPase activity at37 "C reported by Hidalgo et al.( 3 ) . E. Bicknell-Brown and K. G. Brown, unpublished results.

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Composition SR Membrane

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RESULTS

TABLEI1 Cholesterol content of lipids of various SR _ _ _ Source of Measurement Deviations-Error introduced by ". _____ Extra-annuNumber procedures was estimated asfollows. Standards preparedfrom Phospholipid/ phuslar" Muscle type synthetic phosphatidylcholine and synthetic phosphatidylethcholesterol" pholipid/ "f ra"b'ts cholesterol anolamine were compared to inorganic phosphate standards _" molar ratro molar/ratro and gave essentially identical standard curves. Standard deviations for phosphate and protein measurements of a given Combined (back, hind 8 6.8 7.7 +- 2.8 legs) muscle-type sample from a single rabbit were estimated by 10.4 7 12.0 f 2.2 measuring the concentrationsof five dilutions of each sample. Caudofemoralis (fast twitch) The standard deviationfor the five measurements for single 3.1 5 6.0 f 3.5 Soleus (slow twitch) samples averaged 9% of the mean values, both in the case of (mixed Tibialus 11.1 6.2 5.8 6 protein measurements andin the case of phosphate measuretwitch) ___ ments. Standard deviation for cholesterol measurements was ' Calculated by assuming cholesterol is excluded from the annulus estimated from measurements of three dilutions of each sam- (23), which contains 30 phospholipids/Ca'"-ATPase (24). ple and was found to average 220% of the mean. The mean Standard deviation represents differences between rabbits except deviation determined for any one analysis of one rabbit SR for the caudofemoralis where the standard deviationis within expersample is much less than the deviation from the mean for all imental error. rabbits, with the exception of per cent PCin combined muscle and cholesterol in caudofemoralis. Thus, the mean deviations Assuming that the ratio of annular lipids to Ca2+-ATPaseis presented in Tables 1-111 can generally be attributed to vari- 30:1 as determinedby Hesketh etal. (24), and thatcholesterol ations from rabbit to rabbit. is excluded from the annulus, we also calculated the percentSR Phospholipid/Protein Molar Ratios-For each muscle- age in the bulk lipid for each muscle type for each rabbit. The SR, the molar ratio of average overall for rabbits was found to be 10% for caudofetype SR and forcombinedmuscle phospholipid to Ca"-ATPase was determined. Forcalculating moralis SR and 33% for soleus SR (see Table 11). In this moles of Ca"-ATPase, the reported M, = 102,000 for Ca"calculation, the bulk lipid cholesterol is more greatlyenriched ATPase (1) was used. Moles of phospholipid were obtained for soleus SR than caudofemoralis SR because of the generally from moles of lipid phosphate, as described above.The results, lower lipid/ATPase molar ratios found in soleus SH memdisplayed in Table I, include standard deviations which rep- brane samples. resent variations in individual rabbits from the averagevalue Phospholipid Composition-The average phospholipid for the number of rabbits studied. A broad range of values, compositions determined for each of the various types of SR attributed todifferences inrabbits, is observed for each muscle are presented in Table 111. Significant differences in PC, SM, type, as evidenced by the large deviations from the mean and PI are evidentamong the differing types of SR. values. Because of the large deviations, it is difficult to attach The percentagesof phosphatidylcholine for caudofemoralis any significance to differences in mean values for different and for tibialus SR membranesarecomparabletothose muscle types. We can tentatively speculate that the lipid/ observed for combined muscle SR. However, the percentage ATPase molar ratio values are more frequentlyin the lower PC in soleus SR is about half (0.59) that of caudofemoralis, range for soleus than for the other muscle types. for example. Althoughthe variation among rabbits and, hence, Cholesterol Content for SR of Different Muscle Types-For the standard deviations arelarge, per cent PC was much less the different muscle types, themolar ratios of phospholipid to in soleus SR than in other typesof SR for all rabbits used. T o cholesterol are presented in Table 11. Per cent cholesterol illustrate this point, we calculated for each rabbit the ratioof compositions for the SR lipids are about 8% for caudofemor- the per cent PC in caudofemoralis to the per cent PC in soleus alis, 17% for soleus, 9% for tibialus, and 13% for combined and averaged the comparative ratiofor all rabbits. The avermuscles. The average per centcholesterol composition of the age comparative ratio, with the standard deviation for the lipid was half as great for caudofemoralis SR (representative ratio, presented in Table IV, is 1.7. of fast twitch muscle) as for soleus SR (representative of slow The decreased per cent PC in soleus SR relative to other twitch muscle). Although the standard deviations are large types of SR appears to be coupled to increased per cent SM. because of differences in rabbits, per cent cholesterol compo- Per cent SM is about 12 in soleus SR and 4-5 in other types sition of caudofemoralis (fast twitch) SR was about half that of SR. Thus, the total choline head group is about 60%' for of soleus (slow twitch) S R in every rabbitanalyzed. The ratio both soleus and caudofemoralis. Such coupling has been obof cholesterol composition in caudofemoralis to that in soleus served in other systems (25).It is interesting to note that the was calculated for each rabbit. The mean value of 0.45 for this per cent PC and SMin the soleus showed the greatest variaratio(Table IV)clearly statesthat cholesterol content is tion from rabbit to rabbit, suggesting that variationin PC and significantly less in caudofemoralis SR than soleus SR. Evi- SM content may have a regulatory role in soleus muscle SR. dence that cholesterol is excluded from the annulus of SR Because of the large deviation among rabbits, it is again Ca"-ATPase (20) would suggest that the per centcholesterol much easier to see that the increased per cent SMin soleus is in the bulk lipid may differ fromthat of the total lipid. real if we look at the average"comparative ratio" (Table IV). The ratio of the per cent SM in caudofemoralis to per cent TABLEI SM in soleus was calculated for each rabbit and averaged to Molar " ratio various types SK _~ " " " _ phospholipid to Cn'"-ATPa.se for -_____ obtain the average comparative ratio, which is 0.34. This value Phospholipid/Ca-"1, indicating that. theper cent SM is is clearly much less than Muscle Lype ATPase" indeed greater in soleus than caudofemoralis. analyzed The average per cent PIvalue for soleus SR was about 2.5 molnr rntlo times greater than thatfor caudofemoralis SR. However, the Combined (hack, hind legs) 257 f 60 9 228 f 51 9 Caudofemoralis (fast twitch) per cent PI in the soleus SR was much greater than that of 200 f 86 7 Soleus (slow twitch) caudofemoralis SR in three of the four rabbits from which Tibialus 325 f 146 7 sufficient quantities of both types SR were obtained, but was ______"_~."______~ Standard deviation represents differences between rabbits. not detected in the soleus of the fourth rabbit. For the three "~

*

:kts

I'

"

Composition SR Membrane

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TABLE I11 Phospholipid head group composition Number

Muscle type PC

PE"

PS

I

I

%,

Combined (back, 168 f 6 116 f 3 7 f 2 hind legs) Caudofemoralis (fast twitch) Soleus (slow twitch) 37 f 22 33 f 8 10 f 3 Tibialus 7 410 f9f 41f 21 Literature values for combined muscles Range

65-71

12-19

0-9.2

68f3 16f3 5 f 4 Average '' PE, phosphatidylethanolamine; PS, phosphatidylserine. Ref. 8-13.

TABLE IV Comparison of caudofemoralis and soleus SR Composition measurement

Comparative ratio: 3 ' composition value for caudofemoralis ratioed to I composition value for soleus

molar ratio

Cholesterol to phospholipid ATPase Cholesterol to PC to total phospholipid phospholipid" total to PI SM to total phospholipid For three of six rabbits studied(see text).

0.45 f 0.27 0.32 f 0.19 1.7 f 0.7 0.17 f 0.1 0.34 f 0.36

rabbits, the average of the comparative ratio of caudofemoralis per cent PI tosoleus per cent PIwas 0.17, which is much less than 1 (Table IV). The composition we determined for our combined muscle SR is comparable to the values previously reported (Table 111) for SR composition of combined muscles (8-13). Cardiolipin and phosphatidic acid, which are not reported in Table 111, wereobservedin traceamounts inall types SR we (8-11) for combined analyzed, whichagrees with other reports muscles.

SR membrane to rigidify the bilayer and decrease ATPase activity. We believe the lower PC composition observed for slow decreased ATPase activity twitch soleusSR may contribute to and slower muscle relaxation.Warren et al. ( 2 3 )have observed that dipalmitoylphosphatidylcholine inhibits Ca"-ATPase activity, while Knowles et aZ. (31) have observed that PC stimulates Ca2'-ATPase activity. Knowles and Racker (32) later found that PCin the presence of phosphatidylethanolamine acts synergistically to stimulate Ca"-ATPase activity. We expect the lower sphingomyelin content of slow twitch soleus SR toalso contribute to decreasedCa'+-ATPase activity and slower muscle relaxation. The variable acyl hydrocarbon chain of S M is found to be highly saturated in natural membranes (25). According to Lentz et al. (33), varying the SM to PC ratio maybea means of regulatingside chain saturation and, hence, bilayer fluidity (34). Thus, we expect higher SM/PC ratios in slow twitch soleus SR to contribute to increased bilayer rigidity and decreased ATPase activity. PI, which is observed in less abundance in slow twitch SR, has been linked with nerve impulse propagation at the nerve cell axon (34). However, we are stillin doubtastothe significance of decreased levels of PI in soleus SR. The differences inlipid composition for slow twitch andfast twitch SR are totally consistentwith differences in the properties of the two SR types. It is remarkable that, for each difference in soleus and caudofemoralis muscle composition, the predicted effect on Cas'-ATPase activity agrees with the observed difference in Ca2+-ATPaseactivity. Therefore, we propose that the lipid and cholesterol compositions of the sarcoplasmic reticulum aresignificant in regulating differences in Ca2'-ATPase activities andmuscle relaxation rates for slow and fast twitchmuscle. Acknowledgments-We appreciate the technical assistance of Joan Cameron and Marla Applehaum in preparation of the manuscript.

REFERENCES

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