THE 246, No. JOURNAL 18, Issue cm BIOLOGICAL of September Printed CHEMISTRY 25, pp. Vol. 5617-5620, 1971 in U.S.A. Lipids and Mitochondria and Fatty Acids of Sarcolemma, from Sarcoplasmic Muscle* Reticulum, Rat Skeletal (Received for publication, May 4, 1971) W. FIEHN~ AND J.B. From the Department of Medicine, UCLA Xchool of Medicine, Los Angeles, California 90024 PETER JAMES F. MEAD M. GAP-ELEPANO AND From the Institute for Nuclear 90024 Medicine and Racliobiology, University of California, Los Angeles, California Downloaded from SUMMARY of neutral calcium accumulation Recent studies unsaturated the calcium transport Although membranes characteristic For example, Keenan and Morrb branes of liver cells with composition (7). We recently reported highly purified ATPase could be demonstrated by electron microscopy, fragmented sarcoplasmic this paper we will show distinct acid composition but leaves the ATPase (4, 5) also emphasized fatty acids in the beta position ATPase the lipid and especially the fatty acid composition varies considerably from species and with features of certain membranes (7) distinguished regard to their unimpaired (4, 5) of the the importance of phospholipids 1. The pholipids sarcoplasmic muscle. 2. The different chondria. phosphatidylserine well as large fatty acids in the neutral 3. The fatty acid content 16:0 + 18:0 chondria or fragmented very similar. fatty acid pattern major were classes determined reticulum, lipids and the fragmented of rat phos- in in mitochondria sarcolemma, of FSR. and skeletal of diet (6), lipid from The composition that sarcolemma and sphingomyelin amounts of cholesterol, of the sarcolemma reticulum a high in the phospholipid cholesterol is clearly and content http://www.jbc.org/ are being recognized. various mem- lipid and fatty acid of sarcoplasmic mito- contains of as ester and from skeletal muscle of an active (Na+ The preparation, is free of contamination reticulum (FSR) or mitochondria. differences in the lipid and fatty of sarcolemma, FSR, and mitochondria. the preparation in which lipid fraction. of the neutral sarcolemma + K+)- as judged by guest on May 4, 2020 lipids is high of mito- which a distinct in (8). and is less unsaturated sarcoplasmic Each of the cell fractions in the phospholipid than that by recognizable reticulum, are In shows fraction. METHODS Mature on Purina lab chow were used for all studies. plasmic reticulum (sarcotubular scribed by Hasselbach and sarcolemma (8) according Lipid extraction and purification those of Folch et al. (11) and Sperry matography with quantification according to the method of Fewster, Burns, and Mead Neutral lipids and phospholipids acid column with chloroform Methyl esters were obtained methanolic HCI for 2 hours at 85” in closed vessels. chromatography was performed Standards obtained from Hormel were used for identification tography. Where standards were not available esters were identified by their relative retention to the method of Hofstetter, cation was done by triangulation composition of the fatty acid mixtures male Wistar rats weighing about 200 g and maintained Fragmented vesicles) was prepared and Makinose (9) and mitochondria to the methods of Peter. methods (lla). by densitometry sarco- as de- (10) Although membranes, it is established dria (1) or fragmented either activity or both. This is especially and its associated ATPase, when the phospholipids or De2 By contrast, phospholipase lipids into lysolecithin the interaction the proteins and the lipids, is not clearly understood, that disruption of this interaction sarcoplasmic of certain enzymes or active transport clear for calcium which are both abolished are hydrolyzed A digestion and unsaturated between the main constituents of were essentially Thin layer chro- was performed (12). from a silicic respectively. with Gas liquid 7400 apparatus. Austin, Minnesota, in mitochon- (2-5) processes, transport completely by phospholipase of FSR phospho- fatty acids abolishes reticulum inhibits in FSRl were eluted and methanol, by transmethylation C (2,3) 6% with a Packard Institute, in thin layer and gas liquid * This study was supported Service Grant NS 07587. $ Paul Cohen Postdoctoral Associations of America. 1 The abbreviations sarcoplasmic reticulum; SL, sarcolemma. 2 W. Fiehn and W. Hasselbach, by United States Public Health chroma- Fellow of the Muscular Dystrophy fatty acid methyl times according (13). Quantifi- used are: FA, fatty acids; FSR, fragmented NL, neutral lipids; Sen, and Holman of the peaks in both cases. is estimated PL, phospholipids; The submitted for publication. from the 5617
5618 Lipids of Xubcellular Fractions of Muscle Vol. 246, No. 18 I TABLE Lipid three FSR, and three mitochondrial content of sarcolemma, FSR, and preparations mitochondria of rat skeletal muscle Mean of six sarcolemma, (&S.E.). Mitochondria SL FSR mg lipiafmg g~0th2 1.06 0.51 0.54 mg zipia/mg protein 0.58 f 0.45 0.13 protein 0.02 mg lipid/mg % &.s.E.) % kt:S.E.) % kS.E.1 Total Phospholipids. NL (%) lipids.. . . . . , f 0.16 0.02 0.37 f f 1.1 f 1.15 0.31 0.06 48 f 2.0 78.0 22.0 82.7 17.3 f f 1.7 1.7 51.9 f 2.4 II TABLE Neutral lipid composition and percentage of sarcolemma, FSR, of total neutral and mitochondria Values are milligrams of lipid per mg of protein (+S.E.) lipids. SL Mitochondria FSR mg zipiah protein 0.151 0.161 0.156 0.108 mg lipid/mg protein 0.093 0.010 0.021 0.003 mg ligidfnzg protein 0.053 0.0014 0.007 0.0003 % (fS.E.1 29.9 f 21.5 28.5 18.5 f 0.2 % k!z:S.E.) % e=S.E.) Triglycerides. Free fatty acids. Cholesterol Cholesterol . . . . . . . . . 1.8 3.6 1.7 73.3 7.9 16.2 2.6 f XII 0.5 f 1 f 0.1 3 83.5 2.2 11.5 0.7 f =t f f 0.1 3 0.2 0.8 . . . . . . zk f Downloaded from free.............. esters............ III TABLE Phospholipid composition of sarcolemma, FSR, and mitochondria http://www.jbc.org/ Values are milligrams error of meal1 of lipid per mg of protein preparations and three analyses of each subcellular and y. (&S.E.) of total phospholipid fraction. in each subcellular fraction; S.E., standard - I of two SL Mitochondria FSR % kiz.s E.) 48.9 f 1.1 39.3 f 9.0 zk 2.9 f mg zipia/mg fir&in mng lipidfmg protein % kkS.E.) % kttS.E.) I by guest on May 4, 2020 Phosphatidylcholine Phosphatidylethanolamine Phosphatidylserine. Sphingomyelin. f 1.8 f * f 0.3 0.232 0.114 0.090 0.073 45.5 22.4 17.6 14.4 f f f f 2.8 2.6 0.2 1.5 0.454 0.229 0.073 0.025 58.2 29.4 9.4 3.2 0.404 0.330 0.074 0.024 0.6 0.9 2.3 0.9 0.3 . . . . . - phosphatidylethanolamine all three fractions in SL. In sarcolemma 4-fold and phosphatidylserine 2-fold of that of FSR and mitochondria Significant differences total NL and PL are also present stearic acid are high and the relative linoleic acids are correspondingly sarcolemma in comparison The fatty acid composition differences, especially the larger proportion acids, 22:6, in FSR and mitochondria. unsaturated fatty acids amount mitochondria, and 51% in the sarcolemma; fatty acids are found in amounts chondria, and only 16% in the sarcolemma IV). The dimethyl acetyl content lular particles with the C-16 compound The acetal content was found to be higher in the PL than in the NL fraction (Table V). fraction is the second highest PL in amounts of PL is is almost area of the respective PL fractions saponification methanolic Cholesterol the method of Zlatkis, Zak, and Boyle (14) after thinlayer tography separation with 1,2-dichlorethane. mined by the Lowry method peaks. Dimethyl by gas liquid acetals of the NL and chromatography ester mixture with pentane. esters were also determined were identified of the fatty acid methyl KOH followed and cholesterol after 6% but is present in distinctly sphingomyelin as percentage smaller with as percentage by extraction of PL III). by (Table chroma- was deter- in the fatty acid composition (Table proportions low in the NL with FSR and mitochondria. of the PL fraction of the and Protein as a standard. IV). Palmitic of oleic and fraction (15) with albumin of RESULTS The total lipids per mg of protein lowest in the mitochondria. are both very rich in phospholipids, about 50% of the total extracted I). The major components chondria are triglycerides, ponent of the neutral esterified cholesterol in sarcolemma, about 10% in FSR and 5% in the mitochondria preparations. The high content NL fraction of sarcolemma (Table In all three subcellular fractions choline with the highest content are highest of mitochondria whereas in SL preparations lipids are neutral of the NL fraction of FSR and mito- whereas cholesterol lipids in sarcolemma amounts to 25 to 30% of the total cholesterol in the SL and and FSR also shows large of the polyene fatty In the PL fraction poly- to 67% in the FSR, 64% in the unsaturated of 27% in FSR, 260/, in mito- preparation Lipids lipids (Table C-22 is the main com- (Table II). The (Table was similar for all three subcel- as the main component. of free fatty acids found in the II) is noteworthy. the main PL is phosphatidyl- in FSR (Table III). The
Issue of September 25, 1971 W. Fiehn, J. B. Peter, J. F. Mead, and M. Gan-Elepano 5619 IV studies.3 tally and spectrophotometrically detectable cytochrome The lipid composition with other cell membranes. neutral lipids Morre (7) for the liver plasma membrane, terol content is somewhat somewhat lower. As with liver plasma membrane, content of free fatty acids in sarcolemma. bility cannot be ruled out that fatty acids (FA) were liberated during the long preparation procedure, fatty acids may be truly typical such high FA content was found in the other were subjected to the same procedures. typical of plasma membranes seem to be relatively of sphingomyelin (7, 17) and phosphatidylserine which are high in the phospholipid Patton (18) recently noted cholesterol, sphingomyelin, and total membranes of the rat hepatocyte. relationship and show that the phospholipid creases progressively from mitochondria of protein) through FSR (0.45) to SL (0.51). sive increase occurs in the molar ratios of cholesterol myelin, from 1.6 for mitochondria Complexes between cholesterol portant units of membrane structure. The differences found in the fatty acid patterns pholipids isolated from the three different structures The high unsaturation in the fatty acids of the PL fraction SR and mitochondria is in sharp contrast position of sarcolemma. These differences in the lipid and the fatty acid composition probably reflect the different roles the membranes cell. Thus the plasma membrane, location for several enzymes and enzymatically systems, has an important structural, function. For the latter roles the high might be essential as it has been shown that cholesterol artificial lipid membranes increases molecular lipid orientations by limiting carbon chains of the phospholipids, of the layer as indicated by a decrease of the membrane bility (19, 20). The FSR membrane, on the other hand, is apparently specialized membrane which was shown to be composed mainly of functional protein and phospholipids proportion of “structural” protein and lipids, not involved calcium transport system. The high fatty acids in FSR may reflect the important rated fatty acids in the beta-position transport ATPase of FSR (5). While this paper was in preparation, Sonnenblick (23) studied mitochondria, from dog heart. Their failure to find any striking type and amount of lipid in the various subcellular is difficult to reconcile with our data and those of previous vestigators (7), even considering Both sarcolemma and FSR were studied polarographi- and were found to contain oxidase activity. of sarcolemma shows features in common For example, the composition closely resembles that described TABLE no of of neutral and mitochondria skeletal muscle and phospholipids Fatty acid sarcolemma, composition of rat FSR, of the and Values, expressed as percentage fraction, are the mean of analyses of two preparations; errors for all fatty acids are <lo’% and 18:l (&SE. 18%) of the SL. I of total fatty acids in each by Keenan standard except for 18:0 (&.E. 20%) except that its choles- ester content there is high Although higher and the cholesterol Neutral lipids Phosphoiipids the possi- 7 Mito- chon- dria SL FSR FSR this high content for a plasma membrane. of free No fractions characteristics high amounts (17), both of of sarcolemma. correlation content in different data support content (0.31 mg of PL per mg The most impres- which 14:o ................... 16:O ................... 16:1................... 18:0 ................... 18:l.. ................. 18:2 ................... 20:0 and/or 18: 3~3. ................ 20:3w6 ................. 20:4w6 ................. 20:3 ................... 20:5w3. 22:4w6 ................. 22:5w6 ................. 22:503 ................. 22:6o3 ................. 6.0 27.1 5.8 14.6 21.9 11.6 1.8 0.4 4.0 18.8 6.3 9.1 27.6 24.5 3.2 0.8 4.0 18.2 5.7 7.3 30.5 24.0 2.0 0.3 12.5 0.7 11.9 7.6 14.5 0.5 0.2 0.9 22.6 0.9 1.0 0.6 12.0 1.1 14.8 8.1 21.0 0.1 0.2 0.8 14.4 0.2 0.4 0.3 0.2 6.4 19.9 Other 17.6 2.7 15.2 11.8 11.8 2.7 0.2 2.8 12.8 4.2 1.3 fraction a positive between 18:3w6 ..... Downloaded from lipid Our this also in- 2.5 5.6 1.4 1.5 0.7 2.0 2.0 0.3‘ ............. to sphingo- http://www.jbc.org/ to 3 for FSR and 4.5 for SL. and sphingomyelin 0.5 0.9 1.3 1.5 3.2 11.2 1.0 1.2 3.5 23.2 may be im- - - of the phos- are striking. TABLE V of by guest on May 4, 2020 type of dimethyl subcellular to the fatty acid com- Quantity and acetals present in fractions Dimethyl wet& play in the to being driven transport and insulating cholesterol Percentage total of fatty acids in addition the c-14 1 C-16 1 C-18 % stabilizing, Sarcolemma PL. NL. FSR PL........... NL. Mitochondria PL NL. . content added to of certain of the hydro- with compaction Trace 27.3 53.1 63.6 46.9 9.1 6.4 4.4 the stability the motion presumably 1.7 11.5 63.3 53.8 35.0 34.6 6.0 2.6 permea- 7.3 74.5 54.3 29.1 45.7 5.5 2.3 Trace a highly (21, 22) with a lower in the DISCUSSION content of unsaturated role that unsatu- Sarcoplasmic according tinct membranous chondria available Hotta and Usami (16) and Peter (8) demonstrated inhibited (Na+ + K+)-ATPase which thus fits the general concept of plasma membranes. the results of our lipid analyses, a significant the isolated sarcolemma by mitochondria as was also indicated by electron reticulum, morphology structures. have been studied for the muscle cell membrane, mitochondria, and physiological Although extensively, and sarcolemma are, dis- to their function, of the PL play in the calcium isolated FSR and mito- there are few data despite its importance. Weglicki, FSR, and sarcolemma differences in components Stam, and a ouabain- in skeletal muscle sarcolemma, From in- contamination of differences in methodology. or FSR can be excluded microscopy and biochemical 3 W. Fiehn, J. B. Peter, L. Rusdal, submitted for publication.
Vol. 246, No. 18 Lipids of Xubcellular Fractions of il!.luscle 5620 12. FEWSTER, M. E., BURNS, B. J., AND MEAD, J. F., J. Chroma- REFERENCES 43, 120 (1969). H., Sot., 42,537 A., ZAK, (1953). 0. H., R. J., J. Biol. A&D USAMI, T. K., SKIPSKI, ARCHBALD, 18. PATTON, 19. CHAPMAN. D. A., 20. VAN DEENEN, brane lipids membrane New Jersey, 21. HASSELBACH, 159 (1966). 22. HASSELBACH, 598 (1967). 23. WEGLICKI, E. N., togr., 1. FLEISCHER, BACK, 2. MARTONOSI, Chem., 3. DIEHL, 24, 440 4. BALZER, Naunyn 456 (1968). 5. FIEHN, (1970). 6. VAN MONTFOORT, Biochemical 7. KEENAN, 8. PETER, 9. HASSELBACH, (1963). 10. PETER, 11. FOLCH, F. N., J. Biol. fla. SPERRY, S., BRIERLEY, D. B., J. Biol. A., 243, 61 (1968). F., MAKINOSE, (1965). H., MAKINOSE, Schmiedebergs G., KLOUWEN, Chem., 237, DONLEY, J., H., AND SLAUTTER- 13. HOFSTETTER, Chem. ZLATKIS. 41,486 15. LOWRY, DALL, 16. HoTTA~K., 17. RAY, SEN, N., AND HOLMAN, R. T., J. Amer. Oil 3264 HALPIN, (1962). (1965). AND BOYLE, AND R. A., J. Biol. 14. B., A. J., J. Lab. Clin. Med., M., AND HASSELBACH, W., Fed. Proc., ROSEBROUGH, N. 193, Biochem., BARCLAY, Chem., 29,489 F.. PHILLIPS, Acta, in D. lipophilic Prentice J., FARR, 265 (1951). A. L., AND RAN- Chem., Y., V. J. Biol. Biol., N. Biophys. L. M., M., FIEHN, W., AND HASSELBACH, Exp. W., 260, j. 61,407 M., 5528 (1967). Arch. Pharmakol. Pathol., P., ESSNER, (1969). E., AND F. M., 244, (1970). W., AND HASSELBACH, W., Eur. J. Biochem., 13, 510 S., J. Theor. D.. Biochim. OWENS. M. C., AND WALKER, DEENEN, L. L. M., A,, AND MULDER, problems T. W., AND MORRB, J. B., Biochem. W., DE GIER, J., HOUTSMULLER, E., in A. C. FRAZER Elsevier, J., Biochemistry, Res. Commun., MAKINOSE, M., U. M. (Editor), (1963). 9,19 49,1362 Biochem. I., 183, TOSTESON proteins Hall, 458 (1968). L. and C. (Editor), Mem- of lipids, Amsterdam in molecular Englewood basis Cliffs, of D. (1969). (1970). 339, function, 1968. W., Inc., Biophys. AND 2. 94 AND SERAYDARIAN, K., Biochem. Z., 346, J. B., Biochem. J., AECOLI, Med., 2,179 M., (1968). W., AND ELFVIN, L. G., J. Res., 17, Ultrastr. I., LEES, Chem., Methods MEATH, (1951a). J. A., AND LE BARON, W. J., JR., STAM, Cardiol., A. C., JR., AND SONNENBLICK, 191, 833 Biochem. J. Mol. Cell. 1, 131 (1970). Downloaded from W. M. Anal., 2,83 (1955). http://www.jbc.org/ by guest on May 4, 2020
Lipids and Fatty Acids of Sarcolemma, Sarcoplasmic Reticulum, and Mitochondria from Rat Skeletal Muscle W. Fiehn, J. B. Peter, James F. Mead and M. Gan-Elepano J. Biol. Chem. 1971, 246:5617-5620. Access the most updated version of this article at http://www.jbc.org/content/246/18/5617 Alerts: • When this article is cited • When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts This article cites 0 references, 0 of which can be accessed free at Downloaded from http://www.jbc.org/content/246/18/5617.full.html#ref-list-1 http://www.jbc.org/ by guest on May 4, 2020