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Evidence for the presence of the stearyl-CoA desaturase system in the sarcoplasmic reticulum of rabbit slow muscle
280
Biochimica et Biophysics Acta, 574 (1979) 0 Elsevier/North-Holland Biomedical Press
280-289
BBA 57408
EVIDENCE FOR THE PRESENCE OF THE STEARYL-CoA DESATURASE SYSTEM IN THE SARCOPLASMIC RETICULUM OF RABBIT SLOW MUSCLE
GIOVANNI ALFRED0
SALVIATI, MARGRETH
ROMEO *
BETTO,
SERGIO
SALVATORI
and
Consiglio Nazionale delle Ricerche, Centro di Studio per la Biologia e la Fisiopatologia Muscolare, Istituto di Patologia Generale, Via Loredan 16, I-35100 Padova (Italy) (Received
November
17th,
1978)
Key words: NADH-cytochrome bs reductase; Cytochrome Flavin; (Rabbit slow muscle sarcoplasmic reticulum)
bs; Stearyl-CoA desaturase,
Summary We have shown that the isolated sarcoplasmic reticulum from rabbit slow muscle contains cytochrome b5 which can be reduced via a flavoprotein, with FAD as the prosthetic group. In the presence of NADH and oxygen, these sarcoplasmic reticulum membranes can convert stearyl-CoA to oleyl-CoA, similarly to liver endoplasmic reticulum membranes. However, the stearyl-CoA desaturase system is virtually lacking in fast muscle sarcoplasmic reticulum. The data suggest that these differences between fast and slow twitch muscle may be related to the characteristic fatty acid composition of phospholipids and the function of the sarcoplasmic reticulum.
Introduction Endoplasmic reticulum membranes contain two distinct electron transport systems with separate functions, one using electrons from NADH and the other from NADPH [ 11. Two of the specific components of the NADH-linked electron transport system, (cytochrome b5 and the flavoprotein NADH-cytochrome bs reductase (EC 1.6.2.2)) were shown to be far more ubiquitous membrane constituents of the endoplasmic reticulum of animal cells than are cytochrome P-450 and the respective reductase (EC 1.6.2.4) [2,3]. This differing distribution is consistent with knowledge that the cytochrome b, system plays a role
* To whom correspondence and reprint requests should be addressed.
281
in the NADH- and oxygen-dependent desaturation of both saturated and desaturated fatty acids, and that these metabolic reactions are of more widespread occurrence, and are much less the expression of tissue metabolic specialization, than are those involved in cytochrome P-450-mediated drug and steroid metabolism [2,3]. The stearyl-CoA desaturase system, which has been best characterized in the microsomes of liver and adipose tissue, consists of three enzymes, i.e. the NADH-cytochrome b, reductase, cytochrome bg, and the desaturase acting as the terminal electron acceptor [4 -71. Though it was long believed, since the early observations of Garfinkel [ 21, that the concentrations and activities of the components of the cytochrome bs system are lowest in skeletal muscle, as a characteristic of the tissue, we found that the NADH-cytochrome c reductase activity (rotenone-insensitive) of sarcoplasmic reticulum fragments, which are the equivalent of microsomes from non-muscle tissues, varied considerably according to the type of muscle. Whereas it could be confirmed that the sarcoplasmic reticulum reductase activity, taken as a measure of the activity of NADH-cytochrome b5 reductase-cytochrome bg, is very low in rabbit fast (white) muscles, that did not appear to be true for slow (red) muscles [ 8,9], and there was found to be, in general, a reciprocal relationship between electron transport activity and the activity for Ca”-transport of the isolated sarcoplasmic reticulum, among several muscles investigated [lo]. Since it could be argued that the high level of NADH-cytochrome c reductase activity found in sarcoplasmic reticulum fragments from rabbit slow muscle [ 81 might be accounted for, at least in part, by mitochondrial contaminants, it appeared desirable to investigate more carefully the presence of individual components of the stearyl-CoA desaturase system in such fragments obtained by improved techniques [9]. Materials and Methods New Zealand male adult rabbits were used. The animals were fed a stock laboratory diet and were allowed free access to food and water for all experiments. The combined soleus and semitendinosus were used as representative slow muscles, and the adductor as a fast muscle. The sarcoplasmic reticulum fragments were isolated by differential centrifugation from 10% homogenates in 0.3 M sucrose, essentially as described previously [ 91. A crude sarcoplasmic reticulum fraction, sedimented by centrifuging the mitochondria-free supernatant at 150 000 X g for 60 min, was purified by further centrifuging the fragments on 1.2 M sucrose at 114 000 X g for 60 min. The purified sarcoplasmic reticulum fragments were collected at the 0.3/1.2 M sucrose interface, and were stored at --20°C for l---2 days before use. For cytochrome b5 measurements, the purification procedure involved, as a further step, the extraction of the sarcoplasmic reticulum fragments, as described above, with 0.6 M KCl, in order to remove any interfering myoglobin [ 111. Mitochondria were isolated from the myofibrillar supernatant (650 X g for 10 min) of the muscle homogenates by centrifuging at 6500 Xg for 20 mm. Mitochondrial fragments were obtained by sonication of the mitochondrial suspension in 0.3 M sucrose at 300 W for 15 s, and were isolated by the same procedure used for isolation of the sarcoplasmic reticulum.
282
NADH-potassium ferricyanide and NADH-cytochrome c reductase activities, in the presence and absence of rotenone, were measured spectrophotometrically, as reported previously. [8]. For showing the reactivation of NADHpotassium ferricyanide reductase activity in C02-treated sarcoplasmic reticulum fragments by added FAD, the conditions used were patterned after those of Mueller and Miller [13]. The sarcoplasmic reticulum fragments (about 6 mg protein in 1.5 ml 0.3 M sucrose) were flushed with CO2 at 0°C for 10 min, and were resedimented by centrifugation at 150 000 X g for 60 min. The CO,treated sarcoplasmic reticulum fragments were preincubated at 30°C for 5 min with 40 PM FAD before starting the reaction with NADH. Stearyl-CoA desaturase activity was measured essentially as described by Jones et al. [4]. Alternatively, the desaturase activity was measured spectrophotometrically from the reoxidation of cytochrome b5 at 424 nm, after NADH oxidation, in the presence and in the absence of stearyl-CoA, under conditions adapted from the method of Strittmatter et al. [6]. Cytochrome b, was purified from rabbit liver microsomes according to Spatz and Strittmatter [ 121. About 50 ,ul of sarcoplasmic reticulum fragments in 0.3 M sucrose (100 fig protein) were mixed with 2 ~1 of 4% Triton X-100 and incubated for 10 min at 0°C. 20 ~1 of cytochrome b5 solution (1 nmol) were then added and incubation was continued at 0°C for additional 10 min. The mixture was brought to a final volume of 0.4 ml by diluting with 0.02 M Tris-acetate buffer, pH 8.1, and the reaction was started by adding 2 J 1 mM NADH. The reduction of cytochrome b5 and the blank rate of reoxidation of cytochrome b5, after the exhaustion of the NADH, were recorded in a dual-beam Perkin-Elmer spectrophotometer in the presence of 1.0 mM sodium azide. NADH was again added after the addition of 4 ~1 of 1 mM stearyl-CoA and the time-course of cytochrome b5 reduction and reoxidation were again recorded. Cytochrome b5 was usually determined by differential spectrometry with dithionite, under the conditions described by Smuckler et al. [ 141, or with NADH according to Sottocasa et al. [15], as explained in the legend to Fig. 1. The amount of cytochrome b5 was calculated using a difference extinction nm [16]. Myoglobin was coefficient of 185 000 M-l . cm-l at 424-409 measured by the method of Reynafarje [ 171. FAD was measured fluorimetrically as described by Burch [18], on trichloroacetic acid extracts of sarcoplasmic reticulum membranes. SDS-gel electrophoresis on 10% polyacrylamide gels was carried out according to Weber and Osborn [ 191. Protein was determined by the method of Lowry et al. [20]. Results and Discussion 1. Cytochrome
b5
Differential spectrometry (with NADH) of the isolated sarcoplasmic reticulum from rabbit slow muscle, revealed a typical cytochrome b5 spectrum (cf. Strittmatter [3]), with a 409 nm minimum, a 424 nm maximum, and two smaller absorption peaks at 526 nm and 556 nm (Fig. 1). The adding of dithionite, subsequent to NADH, resulted in further reduction of cytochrome b5, as shown by the increase in the extinction difference between 424 and 409 nm (Fig. l), without producing any marked qualitative change in the difference
283
c 400
4
450
500
550
600
Wavelength,nm Fig. 1. Difference spectra of isolated sarcoplasmic reticulum fragments from slow muscle purified as described in Materials and Methods. Each cuvette contained, in a final volume of 0.4 ml. about 0.4 mg of sarcoplasmic reticulum protein. 0.05% deoxycholate and 50 mM potassium phosphate buffer, PH 7.5. 3 mM succinate and 1.5 PM rotenone were added to both cuvettes (trace A), followed by the addition of 60 PM NADH (trace B). and subsequently, of 0.05% sodium dithionite (trace C), to one of the cuvettes.
spectrum, except for the appearance of a small shoulder at about 443 nm. That was probably due to the presence of very low amounts of cytochrome oxidase (which is known to be reducible by dithionite [21], as the result of contamination of the isolated sarcoplasmic reticulum with fragments of the inner mitochondrial membrane [ 91. However, from these data, as well as from measurements of additional mitochondria markers in the isolated sarcoplasmic reticulum, the extent of mitochondrial contamination appeared to be too slight to interfere seriously with determination of cytochrome b5 by differential spectrometry. Thus, firstly the sarcoplasmic reticulum fragments had a succinate-cytochrome c-reductase activity of about 20 nmol cytochrome c reduced per min per mg protein, as compared with an activity of about 200 nmol cytochrome c reduced per min per mg protein found in mitochondrial fragments with identical sedimentation properties (see Materials and Methods). By comparison, Meissner and Fleischer [ 221 reported a succinate-cytochrome c-reductase activity of 5-10 nmol cytochrome c reduced per min per mg protein for highly purified sarcoplasmic reticulum preparations from rabbit fast muscle. Secondly, electrophoresis in sodium dodecyl sulfate polyacrylamide gels of sarcoplasmic reticulum fragments and of mitochondrial fragments of rabbit slow muscle (Fig. 2), revealed different peptide patterns, as well as a certain degree of cross-
284
a
b
C
Fig. 2. Co-electrophoresis of sarcoplasmic reticulum fragments and mitochondrial fragments from rabbit slow skeletal muscle. a, sarcoplasmic reticulum fragments, 8 pg of protein; b, mitochondrial fragments, 8 pg of protein; c. sarcoplasmic reticulum fragments (8 L(g) + mitochondrial fragments (8 pg). The sarcoplasmic reticulum fragments and the mitochondrial fragments were isolated as reported in Materials and Methods and were further purified with cholate and 0.05 M KC1 according to Meissner et al. [281. SDS-gel el&trophoresis was carried out on 10% polyacrslamide gels by the method of Weber and Osbom [191.
contamination between these fractions. However, a doublet of peptides in the 50 000 -60 000 dalton range and a peptide of a molecular weight slightly below 30 000, which were found to be the main components of the mitochondrial fragments, were present only in trace amounts in the sarcoplasmic reticulum fragments. Thirdly, in another study (unpublished results) we have found that cardiolipin, which is a major phospholipid component of mitochondria, of the inner membrane in particular [23], accounted for 0.5% and 10.9%, respectively, of total membrane phospholipids in the sarcoplasmic reticulum fragments and in the isolated mitochondria from slow muscle. Again, our own values for the percentage content of cardiolipin in sarcoplasmic reticulum phospholipids from slow muscle are only about twice those reported by Meissner and Fleischer [ 221 for the isolated sarcoplasmic reticulum from rabbit fast muscle. Also, similarly to the results of Meissner and Fleischer [22], monoaminooxidase, a marker of the outer mitochondrial membrane [24], measured according to Schnaitman [24], was undetectable in our sarcoplasmic reticulum preparations. Furthermore, it was ascertained by using Reynafarje method [17], as well as by differential spectrometry (CO-treated versus untreated) of dithionitereduced sarcoplasmic reticulum fragments, that the extraction with 0.6 M KC1 solution (see Methods) was effective in removing low amounts of contaminant myoglobin (about 30 pg/mg sarcoplasmic reticulum protein) present in these fragments before the final purification step. Accordingly, both myoglobin and hemoglobin were undetectable in the KCl-extracted sarcoplasmic reticulum fragments by the same methods.
Using dithionite as the reducing agent, we found that the isolated sarcoplasmic reticulum from slow muscle contained 0.27 nmol of cytochrome bs per mg protein, on average, i.e. about 25% of the reported values for rabbit liver microsomes [ 251. On the other hand, only trace amounts of cytochrome bS were found to be present in the isolated sarcoplasmic reticulum from rabbit fast muscle (about 0.01 nmol per mg protein), which is in agreement with the previous studies of Meissner and Fleischer [ 221.
Fluorometric analysis of trichloroacetic acid extracts of the sarcoplasmic reticulum membranes from slow muscle showed a very low content of FMN (0.04 nmol per mg protein) and a high content of FAD (0.24 nmol per mg protein) i.e. a flavin composition similar to that of liver microsomal membranes 1261, and different from that of submitochondri~ particles endowed with NADH-cytochrome c reductase activity, where the acid-extractable flavin is mostly FMN [ 271. In confirmation of our own earlier results [S], the NADH-cytochrome c reductase activity of the isolated sarcoplasmic reticulum appeared to be largely insensitive to rotenone, and measured in its presence, had mean values of 1.15 ymol cytochrome c reduced per min per mg protein, which are similar to those reported for liver microsomes [ 151. That electron flow from NADH to cytochrome c, in the isolated sarcoplasmic reticulum from slow muscle, occurs mainly through the flavoprotein NADH-cytochrome 6, reductase and cytochrome bS, and that these two proteins are truly constituents of the sarcoplasmic reticulum membranes, is further supported by the following experimental evidence. Partial extraction of the isolated sarcoplasmic reticulum with low concentrations of cholate, in the presence of 0.5 M KCl, i.e. under the conditions described by Meissner et al. for purifying the sarcoreticular ATPase [28], resulted in a decrease of membrane-bound NADH-cytochrome e reductase activity of the reisolated vesicles to 0.11~01 cytochrome c reduced per min per mg protein, and in a concomitant release to the supernatant of 85% of the cytochrome b, originally present. Therefore, these results are consistent with knowledge that both cytochrome b5 and the respective reductase are detached from microsomal membranes by mild detergent treatment [ 11. Furthermore, it is well known both that NADH-cytochrome b5 reductase has FAD as the prosthetic group [29] and that potassium ferricyanide acts as a direct electron acceptor from the reduced flavoprotein [29]. Activity measurements of this flavoprotein enzyme from the rate of reduction of potassium ferricyanide by added NADH are, however, complicated in the presence of even low amounts of mitochondrial contaminants, since potassium ferricyanide is an efficient electron acceptor for the NADH-cytochrome c reduetase of submitoehondrial particles 1273. We tried to distin~ish between these two different activities by dissociating the prosthetic group from NADH-cytochrome b5 reductase, and following the decrease in total NADH-potassium ferricyanide reductase activity of the sarcoplasmic reticulum fragments, and its subsequent reactivation by added FAD. It was found that when the sarcoplasmic reticulum fragments were subjected to mild acid treatment, NADH-potassium ferricyanide
286
reductase activity decreased from 3.72 pmol potassium ferricyanide reduced per min per mg protein to 1.53 pmol, and was restored almost completely to the original value by added FAD (3.42 pmol/min per mg protein). Interestingly, the calculated fraction of the total NADH-potassium ferricyanide reductase activity present in the sarcoplasmic reticulum fragments that could be specifically regained by adding FAD corresponded to values of about 2 pmol potassium ferricyanide reduced per min per mg protein, i.e. similar to the reported values for the NADH-potassium ferricyanide reductase activity of liver microsomes [ 151. 3. Stearyl-CoA desaturase Direct evidence for the presence of stearyl-CoA desaturase activity in the isolated sarcoplasmic reticulum from rabbit slow muscle was obtained in further experiments. The desaturase activity was found to be proportional to the amount of membrane protein added (Fig. 3) and to incubation time (Fig. 4). Under an identical set of experimental conditions, there was virtually no activity in the isolated sarcoplasmic reticulum from rabbit fast muscle. The observed rates of conversion of stearyl-CoA to oleyl-CoA in slow muscle sarcoplasmic reticulum from normally fed rabbits, with values of 0.10 f 0.02 nmol/min per mg protein (mean values of six determinations with different rabbits + S.E.), are in the lower range of values reported by Oshino et al. for liver microsomes of fasted rats [5], and about one tenth of the reported values for animals ‘induced’ by feeding a high carbohydrate diet. Results in substantial agreement were obtained by measuring spectrophotometrically the rate of desaturation of stearyl-CoA from the rate of reoxidation of exogenous deter-
0.3
‘;; 0,
0.2
E -5. al z s 0.1 0
0
0
0.1
0.2
0.3
0.4
SR Protein (mg)
Fig. 3. Effect of increasing protein concentration on the reticulum (SR) fragments from rabbit slow muscle. The tained 50 mM potassium phosphate buffer, pH 7.2, 1 (specific activity 50.4 Ci/mol) and the specified amounts The amount of [l-l 4Clole~l-CoA formed was determined
stearyl-CoA desaturase activity of sarcoplasmic assay medium. in a final volume of 0.5 ml, conmM NADH, 4.06 nmol of [l-14Clstearyl-CoA of protein. Incubation was at 37’C for 10 min. as described by Jones et al. [41.
0.3
z ,o
0.2
E 0 r $
0.1
0 ”
a
1”
lime
20
15
Onin)
Fig. 4. Effect of increasing time of incubation on the stearyl-CoA desaturase reticulum fragments from slow muscle. The desaturase activity was determined of sarcoplasmic reticulum protein.
activity of sarcoplasmic as in Fig. 3, with 200 pg
gent-cytochrome b5 incorporated into reconstructed vesicles after solubilization with Triton X-100, as described in Methods. In the cytochrome b,-enriched SR membranes, upon addition of NADH (Fig. 5), the reduction of cytochrome b5 by endogenous cytochrome b5 reductase proceeded at a very fast rate and a steady-state level of cytochrome b5 reduction was attained in about two minutes, corresponding to about 70% of the cytochrome b5 added. The addition of the stearyl-CoA both shortened the duration of the newly established
a
e
,
J
n 2 min
m 0
d II
:
\
I
Fig. 5. Effect of stearyl-CoA on steady-state reduction level and rate of reoxidation of cytochrome bg. The assay conditions were as described in Methods. After preincubation at 30°C for 5 min. NADH (2 nmol), stearyl-CoA (4 nmol) and again NADH (2 nmol) were added, at the times indicated by arrows (right to left). Changes in absorbance were recorded at 424 nm.
288
steady-state and accelerated the subsequent reoxidation of cytochrome b, by 30%. The rate of oleyl-CoA formed, calculated from the increment in the rate of reoxidation of cytochrome b5 following the addition of substrate, was 0.16 nmol/min per mg protein. By comparison, the stearyl-CoA desaturase activity of rabbit liver microsomes measured under the same conditions was found to be 0.64 nmol/min per mg protein. These several results therefore indicate that in rabbit slow muscle sarcoplasmic reticulum, as in the case of the microsomes from rat liver and adipose tissue [5], the activity of the cytochrome b,-linked desaturase system is ratelimited by the activity of the desaturase. Consequently our present values may represent minimal figures of the potential capacity of rabbit slow-muscle sarcoplasmic reticulum to catalyze the desaturation of stearyl-CoA to oleyl-CoA. Furthermore, it will have to be investigated whether slow-muscle sarcoplasmic reticulum is able to carry out the oxidative formation of polyunsaturated fatty acids, i.e. A6 desaturation, which is likewise believed to involve cytochrome OS [ 301, In this regard, it is worthy to mention that we were recently able to show that the extracted phospholipids of slow-muscle sarcoplasmic reticulum are richer in arachidonic acid and have a higher arachidonic to oleic ratio, as compared with the phospholipids of fast-muscle sarcoplasmic reticulum (unpublished results). The cytochrome 6, electron-transport system, by influencing the fatty acid composition of sarcoplasmic reticulum phospholipids could therefore exert a role on the activity of enzyme proteins which are integral part of the sarcoplasmic reticulum membranes structure, such as the ATPase for Ca*‘or on the membrane permeability transport, and other intrinsic proteins, characteristics [ 311. Alternatively, the presence of the stearyl-CoA desaturase system in the isolated sarcoplasmic reticulum from rabbit slow muscle may reflect the existence of areas of specialization in the intact sarcoplasmic reticulum of this muscle type, as suggested previously by us [9], and could therefore be functionally unrelated to the Ca*‘-transport activity of the sarcoplasmic reticulum membranes. Acknowledgements This work was supported in part by a grant from Muscular Dystrophy Association. The skillful technical assistance of Mr. Renato Siligardi is gratefully acknowledged. We also would like to thank Professor Lars Ernster for helpful advice in the course of this investigation. References 1
De Pierre,
J.W.
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A., SaIviati, G. and Catani. C. (1971) Arch. Biochem. Biophys. 144, 768-772 A., Salviati, G. and Salvatori. S. (1977) in Membranous Elements and Movement
cules (Reid, E., ed.), Vol. 6, PP. 26-37, Ellis Horwood, Chichester, U.K. 10 Margreth, A., SaIviati, G., Carraro, U. and Mussini, I. (1974) in Calcium
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27 28 29 30 31
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of Mole(Drabi-
kowski. V., Strzelecka-Golaszewska, H. and CarafoIi, E., eds.). PP. 519-545, PWN-Polish Scientific Publishers, Warsaw, and Elsevier, Amsterdam Strittmatter, C.F. (1963) Arch. Biochem. Biophys. 102, 293-305 Spatz, L. and Strittmatter, P. (1971) Proc. Natl. Acad. Sci. U.S. 68, 1042-1046 Mueller, G.C. and Miller. J.A. (1950) J. Biol. Chem. 185,145-154 Smuckler, E.A., Arrhenius, E. and Hultin, T. (1967) Biochem. J. 103. 55-l Sottocasa. G.L.. Kuylenstiema, B.. Em&r, L. and Bergstrand, A. (1967) J. Cell Biol. 32, 415-438 Strittmatter, C.F. and ToIIey Umberger. F. (1969) Biochim. Biophys. Acta 180,18-23 Reynafarje. B. (1963) J. Lab. Clin. Med. 61. 138-143 Burch, H.B. (1957) Methods Enzymol. 3,96*962 Weber, K. and Osbom, M. (1969) J. Biol. Chem. 244, 4406-4410 Lowry, O.H., Rosebrough. N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193. 265-275 Takashi. Y. (1967) Methods Enzymol. 10, 332-335 Meissner, G. and Fleischer, S. (1971) Biochim. Biophys. Acta 241, 356-378 Comte. J., Maisterrena, B. and Gautberon, D.C. (1976) Biochim. Biophys. Acta 419, 271-284 Schnaitmsn. C., Erwin. U.G. and GreenawaIt. J.W. (1967) J. Cell Biol. 32.719-735 Mathews, F.S. and Czerwinski. E.W. (1976) in The Enzymes of Biological Membranes (Martonosi, A., ed.). Vol. 4, pp. 143-197, Wiley, New York Estabrook, R.W., Wersingloer, J.. Masters, B.S.S.. Jonen, H.. Matsubara. T. Ebel. R.. O’Keeffe, D. and Peterson, J.A. (1976) in The Structural Basis of Membrane Function (Hatefi. Y. and DjavadiOhaniance, L., eds.). PP. 429-445, Academic Press, New York Hatefi, Y. and Rieske, J.S. (1967) Methods Enzymol. 10. 225-231 Meissner, G., Conner, G.E. and Fleischer, S. (1973) Biochim. Biophys. Acta 298. 269-276 Strittmatter. P. (1967) Methods Enzymol. 10, 561-565 Brenner, R.R. (1977) in Experimental Medicine and Biology (Brazan, N.G., Brenner. R.R. and Giusto, N.M.. eds.), Vol. 83, pp. 85-101. Plenum Press, New York Boland, R.L. and Martonosi, A. (1977) in Experimental Medicine and Biology (Bazan, N.G., Brenner, R.R. and Giusto, N.M., eds.), Vol. 83, pp. 233-239. Plenum Press. New York
Biochimica et Biophysics Acta, 574 (1979) 0 Elsevier/North-Holland Biomedical Press
280-289
BBA 57408
EVIDENCE FOR THE PRESENCE OF THE STEARYL-CoA DESATURASE SYSTEM IN THE SARCOPLASMIC RETICULUM OF RABBIT SLOW MUSCLE
GIOVANNI ALFRED0
SALVIATI, MARGRETH
ROMEO *
BETTO,
SERGIO
SALVATORI
and
Consiglio Nazionale delle Ricerche, Centro di Studio per la Biologia e la Fisiopatologia Muscolare, Istituto di Patologia Generale, Via Loredan 16, I-35100 Padova (Italy) (Received
November
17th,
1978)
Key words: NADH-cytochrome bs reductase; Cytochrome Flavin; (Rabbit slow muscle sarcoplasmic reticulum)
bs; Stearyl-CoA desaturase,
Summary We have shown that the isolated sarcoplasmic reticulum from rabbit slow muscle contains cytochrome b5 which can be reduced via a flavoprotein, with FAD as the prosthetic group. In the presence of NADH and oxygen, these sarcoplasmic reticulum membranes can convert stearyl-CoA to oleyl-CoA, similarly to liver endoplasmic reticulum membranes. However, the stearyl-CoA desaturase system is virtually lacking in fast muscle sarcoplasmic reticulum. The data suggest that these differences between fast and slow twitch muscle may be related to the characteristic fatty acid composition of phospholipids and the function of the sarcoplasmic reticulum.
Introduction Endoplasmic reticulum membranes contain two distinct electron transport systems with separate functions, one using electrons from NADH and the other from NADPH [ 11. Two of the specific components of the NADH-linked electron transport system, (cytochrome b5 and the flavoprotein NADH-cytochrome bs reductase (EC 1.6.2.2)) were shown to be far more ubiquitous membrane constituents of the endoplasmic reticulum of animal cells than are cytochrome P-450 and the respective reductase (EC 1.6.2.4) [2,3]. This differing distribution is consistent with knowledge that the cytochrome b, system plays a role
* To whom correspondence and reprint requests should be addressed.
281
in the NADH- and oxygen-dependent desaturation of both saturated and desaturated fatty acids, and that these metabolic reactions are of more widespread occurrence, and are much less the expression of tissue metabolic specialization, than are those involved in cytochrome P-450-mediated drug and steroid metabolism [2,3]. The stearyl-CoA desaturase system, which has been best characterized in the microsomes of liver and adipose tissue, consists of three enzymes, i.e. the NADH-cytochrome b, reductase, cytochrome bg, and the desaturase acting as the terminal electron acceptor [4 -71. Though it was long believed, since the early observations of Garfinkel [ 21, that the concentrations and activities of the components of the cytochrome bs system are lowest in skeletal muscle, as a characteristic of the tissue, we found that the NADH-cytochrome c reductase activity (rotenone-insensitive) of sarcoplasmic reticulum fragments, which are the equivalent of microsomes from non-muscle tissues, varied considerably according to the type of muscle. Whereas it could be confirmed that the sarcoplasmic reticulum reductase activity, taken as a measure of the activity of NADH-cytochrome b5 reductase-cytochrome bg, is very low in rabbit fast (white) muscles, that did not appear to be true for slow (red) muscles [ 8,9], and there was found to be, in general, a reciprocal relationship between electron transport activity and the activity for Ca”-transport of the isolated sarcoplasmic reticulum, among several muscles investigated [lo]. Since it could be argued that the high level of NADH-cytochrome c reductase activity found in sarcoplasmic reticulum fragments from rabbit slow muscle [ 81 might be accounted for, at least in part, by mitochondrial contaminants, it appeared desirable to investigate more carefully the presence of individual components of the stearyl-CoA desaturase system in such fragments obtained by improved techniques [9]. Materials and Methods New Zealand male adult rabbits were used. The animals were fed a stock laboratory diet and were allowed free access to food and water for all experiments. The combined soleus and semitendinosus were used as representative slow muscles, and the adductor as a fast muscle. The sarcoplasmic reticulum fragments were isolated by differential centrifugation from 10% homogenates in 0.3 M sucrose, essentially as described previously [ 91. A crude sarcoplasmic reticulum fraction, sedimented by centrifuging the mitochondria-free supernatant at 150 000 X g for 60 min, was purified by further centrifuging the fragments on 1.2 M sucrose at 114 000 X g for 60 min. The purified sarcoplasmic reticulum fragments were collected at the 0.3/1.2 M sucrose interface, and were stored at --20°C for l---2 days before use. For cytochrome b5 measurements, the purification procedure involved, as a further step, the extraction of the sarcoplasmic reticulum fragments, as described above, with 0.6 M KCl, in order to remove any interfering myoglobin [ 111. Mitochondria were isolated from the myofibrillar supernatant (650 X g for 10 min) of the muscle homogenates by centrifuging at 6500 Xg for 20 mm. Mitochondrial fragments were obtained by sonication of the mitochondrial suspension in 0.3 M sucrose at 300 W for 15 s, and were isolated by the same procedure used for isolation of the sarcoplasmic reticulum.
282
NADH-potassium ferricyanide and NADH-cytochrome c reductase activities, in the presence and absence of rotenone, were measured spectrophotometrically, as reported previously. [8]. For showing the reactivation of NADHpotassium ferricyanide reductase activity in C02-treated sarcoplasmic reticulum fragments by added FAD, the conditions used were patterned after those of Mueller and Miller [13]. The sarcoplasmic reticulum fragments (about 6 mg protein in 1.5 ml 0.3 M sucrose) were flushed with CO2 at 0°C for 10 min, and were resedimented by centrifugation at 150 000 X g for 60 min. The CO,treated sarcoplasmic reticulum fragments were preincubated at 30°C for 5 min with 40 PM FAD before starting the reaction with NADH. Stearyl-CoA desaturase activity was measured essentially as described by Jones et al. [4]. Alternatively, the desaturase activity was measured spectrophotometrically from the reoxidation of cytochrome b5 at 424 nm, after NADH oxidation, in the presence and in the absence of stearyl-CoA, under conditions adapted from the method of Strittmatter et al. [6]. Cytochrome b, was purified from rabbit liver microsomes according to Spatz and Strittmatter [ 121. About 50 ,ul of sarcoplasmic reticulum fragments in 0.3 M sucrose (100 fig protein) were mixed with 2 ~1 of 4% Triton X-100 and incubated for 10 min at 0°C. 20 ~1 of cytochrome b5 solution (1 nmol) were then added and incubation was continued at 0°C for additional 10 min. The mixture was brought to a final volume of 0.4 ml by diluting with 0.02 M Tris-acetate buffer, pH 8.1, and the reaction was started by adding 2 J 1 mM NADH. The reduction of cytochrome b5 and the blank rate of reoxidation of cytochrome b5, after the exhaustion of the NADH, were recorded in a dual-beam Perkin-Elmer spectrophotometer in the presence of 1.0 mM sodium azide. NADH was again added after the addition of 4 ~1 of 1 mM stearyl-CoA and the time-course of cytochrome b5 reduction and reoxidation were again recorded. Cytochrome b5 was usually determined by differential spectrometry with dithionite, under the conditions described by Smuckler et al. [ 141, or with NADH according to Sottocasa et al. [15], as explained in the legend to Fig. 1. The amount of cytochrome b5 was calculated using a difference extinction nm [16]. Myoglobin was coefficient of 185 000 M-l . cm-l at 424-409 measured by the method of Reynafarje [ 171. FAD was measured fluorimetrically as described by Burch [18], on trichloroacetic acid extracts of sarcoplasmic reticulum membranes. SDS-gel electrophoresis on 10% polyacrylamide gels was carried out according to Weber and Osborn [ 191. Protein was determined by the method of Lowry et al. [20]. Results and Discussion 1. Cytochrome
b5
Differential spectrometry (with NADH) of the isolated sarcoplasmic reticulum from rabbit slow muscle, revealed a typical cytochrome b5 spectrum (cf. Strittmatter [3]), with a 409 nm minimum, a 424 nm maximum, and two smaller absorption peaks at 526 nm and 556 nm (Fig. 1). The adding of dithionite, subsequent to NADH, resulted in further reduction of cytochrome b5, as shown by the increase in the extinction difference between 424 and 409 nm (Fig. l), without producing any marked qualitative change in the difference
283
c 400
4
450
500
550
600
Wavelength,nm Fig. 1. Difference spectra of isolated sarcoplasmic reticulum fragments from slow muscle purified as described in Materials and Methods. Each cuvette contained, in a final volume of 0.4 ml. about 0.4 mg of sarcoplasmic reticulum protein. 0.05% deoxycholate and 50 mM potassium phosphate buffer, PH 7.5. 3 mM succinate and 1.5 PM rotenone were added to both cuvettes (trace A), followed by the addition of 60 PM NADH (trace B). and subsequently, of 0.05% sodium dithionite (trace C), to one of the cuvettes.
spectrum, except for the appearance of a small shoulder at about 443 nm. That was probably due to the presence of very low amounts of cytochrome oxidase (which is known to be reducible by dithionite [21], as the result of contamination of the isolated sarcoplasmic reticulum with fragments of the inner mitochondrial membrane [ 91. However, from these data, as well as from measurements of additional mitochondria markers in the isolated sarcoplasmic reticulum, the extent of mitochondrial contamination appeared to be too slight to interfere seriously with determination of cytochrome b5 by differential spectrometry. Thus, firstly the sarcoplasmic reticulum fragments had a succinate-cytochrome c-reductase activity of about 20 nmol cytochrome c reduced per min per mg protein, as compared with an activity of about 200 nmol cytochrome c reduced per min per mg protein found in mitochondrial fragments with identical sedimentation properties (see Materials and Methods). By comparison, Meissner and Fleischer [ 221 reported a succinate-cytochrome c-reductase activity of 5-10 nmol cytochrome c reduced per min per mg protein for highly purified sarcoplasmic reticulum preparations from rabbit fast muscle. Secondly, electrophoresis in sodium dodecyl sulfate polyacrylamide gels of sarcoplasmic reticulum fragments and of mitochondrial fragments of rabbit slow muscle (Fig. 2), revealed different peptide patterns, as well as a certain degree of cross-
284
a
b
C
Fig. 2. Co-electrophoresis of sarcoplasmic reticulum fragments and mitochondrial fragments from rabbit slow skeletal muscle. a, sarcoplasmic reticulum fragments, 8 pg of protein; b, mitochondrial fragments, 8 pg of protein; c. sarcoplasmic reticulum fragments (8 L(g) + mitochondrial fragments (8 pg). The sarcoplasmic reticulum fragments and the mitochondrial fragments were isolated as reported in Materials and Methods and were further purified with cholate and 0.05 M KC1 according to Meissner et al. [281. SDS-gel el&trophoresis was carried out on 10% polyacrslamide gels by the method of Weber and Osbom [191.
contamination between these fractions. However, a doublet of peptides in the 50 000 -60 000 dalton range and a peptide of a molecular weight slightly below 30 000, which were found to be the main components of the mitochondrial fragments, were present only in trace amounts in the sarcoplasmic reticulum fragments. Thirdly, in another study (unpublished results) we have found that cardiolipin, which is a major phospholipid component of mitochondria, of the inner membrane in particular [23], accounted for 0.5% and 10.9%, respectively, of total membrane phospholipids in the sarcoplasmic reticulum fragments and in the isolated mitochondria from slow muscle. Again, our own values for the percentage content of cardiolipin in sarcoplasmic reticulum phospholipids from slow muscle are only about twice those reported by Meissner and Fleischer [ 221 for the isolated sarcoplasmic reticulum from rabbit fast muscle. Also, similarly to the results of Meissner and Fleischer [22], monoaminooxidase, a marker of the outer mitochondrial membrane [24], measured according to Schnaitman [24], was undetectable in our sarcoplasmic reticulum preparations. Furthermore, it was ascertained by using Reynafarje method [17], as well as by differential spectrometry (CO-treated versus untreated) of dithionitereduced sarcoplasmic reticulum fragments, that the extraction with 0.6 M KC1 solution (see Methods) was effective in removing low amounts of contaminant myoglobin (about 30 pg/mg sarcoplasmic reticulum protein) present in these fragments before the final purification step. Accordingly, both myoglobin and hemoglobin were undetectable in the KCl-extracted sarcoplasmic reticulum fragments by the same methods.
Using dithionite as the reducing agent, we found that the isolated sarcoplasmic reticulum from slow muscle contained 0.27 nmol of cytochrome bs per mg protein, on average, i.e. about 25% of the reported values for rabbit liver microsomes [ 251. On the other hand, only trace amounts of cytochrome bS were found to be present in the isolated sarcoplasmic reticulum from rabbit fast muscle (about 0.01 nmol per mg protein), which is in agreement with the previous studies of Meissner and Fleischer [ 221.
Fluorometric analysis of trichloroacetic acid extracts of the sarcoplasmic reticulum membranes from slow muscle showed a very low content of FMN (0.04 nmol per mg protein) and a high content of FAD (0.24 nmol per mg protein) i.e. a flavin composition similar to that of liver microsomal membranes 1261, and different from that of submitochondri~ particles endowed with NADH-cytochrome c reductase activity, where the acid-extractable flavin is mostly FMN [ 271. In confirmation of our own earlier results [S], the NADH-cytochrome c reductase activity of the isolated sarcoplasmic reticulum appeared to be largely insensitive to rotenone, and measured in its presence, had mean values of 1.15 ymol cytochrome c reduced per min per mg protein, which are similar to those reported for liver microsomes [ 151. That electron flow from NADH to cytochrome c, in the isolated sarcoplasmic reticulum from slow muscle, occurs mainly through the flavoprotein NADH-cytochrome 6, reductase and cytochrome bS, and that these two proteins are truly constituents of the sarcoplasmic reticulum membranes, is further supported by the following experimental evidence. Partial extraction of the isolated sarcoplasmic reticulum with low concentrations of cholate, in the presence of 0.5 M KCl, i.e. under the conditions described by Meissner et al. for purifying the sarcoreticular ATPase [28], resulted in a decrease of membrane-bound NADH-cytochrome e reductase activity of the reisolated vesicles to 0.11~01 cytochrome c reduced per min per mg protein, and in a concomitant release to the supernatant of 85% of the cytochrome b, originally present. Therefore, these results are consistent with knowledge that both cytochrome b5 and the respective reductase are detached from microsomal membranes by mild detergent treatment [ 11. Furthermore, it is well known both that NADH-cytochrome b5 reductase has FAD as the prosthetic group [29] and that potassium ferricyanide acts as a direct electron acceptor from the reduced flavoprotein [29]. Activity measurements of this flavoprotein enzyme from the rate of reduction of potassium ferricyanide by added NADH are, however, complicated in the presence of even low amounts of mitochondrial contaminants, since potassium ferricyanide is an efficient electron acceptor for the NADH-cytochrome c reduetase of submitoehondrial particles 1273. We tried to distin~ish between these two different activities by dissociating the prosthetic group from NADH-cytochrome b5 reductase, and following the decrease in total NADH-potassium ferricyanide reductase activity of the sarcoplasmic reticulum fragments, and its subsequent reactivation by added FAD. It was found that when the sarcoplasmic reticulum fragments were subjected to mild acid treatment, NADH-potassium ferricyanide
286
reductase activity decreased from 3.72 pmol potassium ferricyanide reduced per min per mg protein to 1.53 pmol, and was restored almost completely to the original value by added FAD (3.42 pmol/min per mg protein). Interestingly, the calculated fraction of the total NADH-potassium ferricyanide reductase activity present in the sarcoplasmic reticulum fragments that could be specifically regained by adding FAD corresponded to values of about 2 pmol potassium ferricyanide reduced per min per mg protein, i.e. similar to the reported values for the NADH-potassium ferricyanide reductase activity of liver microsomes [ 151. 3. Stearyl-CoA desaturase Direct evidence for the presence of stearyl-CoA desaturase activity in the isolated sarcoplasmic reticulum from rabbit slow muscle was obtained in further experiments. The desaturase activity was found to be proportional to the amount of membrane protein added (Fig. 3) and to incubation time (Fig. 4). Under an identical set of experimental conditions, there was virtually no activity in the isolated sarcoplasmic reticulum from rabbit fast muscle. The observed rates of conversion of stearyl-CoA to oleyl-CoA in slow muscle sarcoplasmic reticulum from normally fed rabbits, with values of 0.10 f 0.02 nmol/min per mg protein (mean values of six determinations with different rabbits + S.E.), are in the lower range of values reported by Oshino et al. for liver microsomes of fasted rats [5], and about one tenth of the reported values for animals ‘induced’ by feeding a high carbohydrate diet. Results in substantial agreement were obtained by measuring spectrophotometrically the rate of desaturation of stearyl-CoA from the rate of reoxidation of exogenous deter-
0.3
‘;; 0,
0.2
E -5. al z s 0.1 0
0
0
0.1
0.2
0.3
0.4
SR Protein (mg)
Fig. 3. Effect of increasing protein concentration on the reticulum (SR) fragments from rabbit slow muscle. The tained 50 mM potassium phosphate buffer, pH 7.2, 1 (specific activity 50.4 Ci/mol) and the specified amounts The amount of [l-l 4Clole~l-CoA formed was determined
stearyl-CoA desaturase activity of sarcoplasmic assay medium. in a final volume of 0.5 ml, conmM NADH, 4.06 nmol of [l-14Clstearyl-CoA of protein. Incubation was at 37’C for 10 min. as described by Jones et al. [41.
0.3
z ,o
0.2
E 0 r $
0.1
0 ”
a
1”
lime
20
15
Onin)
Fig. 4. Effect of increasing time of incubation on the stearyl-CoA desaturase reticulum fragments from slow muscle. The desaturase activity was determined of sarcoplasmic reticulum protein.
activity of sarcoplasmic as in Fig. 3, with 200 pg
gent-cytochrome b5 incorporated into reconstructed vesicles after solubilization with Triton X-100, as described in Methods. In the cytochrome b,-enriched SR membranes, upon addition of NADH (Fig. 5), the reduction of cytochrome b5 by endogenous cytochrome b5 reductase proceeded at a very fast rate and a steady-state level of cytochrome b5 reduction was attained in about two minutes, corresponding to about 70% of the cytochrome b5 added. The addition of the stearyl-CoA both shortened the duration of the newly established
a
e
,
J
n 2 min
m 0
d II
:
\
I
Fig. 5. Effect of stearyl-CoA on steady-state reduction level and rate of reoxidation of cytochrome bg. The assay conditions were as described in Methods. After preincubation at 30°C for 5 min. NADH (2 nmol), stearyl-CoA (4 nmol) and again NADH (2 nmol) were added, at the times indicated by arrows (right to left). Changes in absorbance were recorded at 424 nm.
288
steady-state and accelerated the subsequent reoxidation of cytochrome b, by 30%. The rate of oleyl-CoA formed, calculated from the increment in the rate of reoxidation of cytochrome b5 following the addition of substrate, was 0.16 nmol/min per mg protein. By comparison, the stearyl-CoA desaturase activity of rabbit liver microsomes measured under the same conditions was found to be 0.64 nmol/min per mg protein. These several results therefore indicate that in rabbit slow muscle sarcoplasmic reticulum, as in the case of the microsomes from rat liver and adipose tissue [5], the activity of the cytochrome b,-linked desaturase system is ratelimited by the activity of the desaturase. Consequently our present values may represent minimal figures of the potential capacity of rabbit slow-muscle sarcoplasmic reticulum to catalyze the desaturation of stearyl-CoA to oleyl-CoA. Furthermore, it will have to be investigated whether slow-muscle sarcoplasmic reticulum is able to carry out the oxidative formation of polyunsaturated fatty acids, i.e. A6 desaturation, which is likewise believed to involve cytochrome OS [ 301, In this regard, it is worthy to mention that we were recently able to show that the extracted phospholipids of slow-muscle sarcoplasmic reticulum are richer in arachidonic acid and have a higher arachidonic to oleic ratio, as compared with the phospholipids of fast-muscle sarcoplasmic reticulum (unpublished results). The cytochrome 6, electron-transport system, by influencing the fatty acid composition of sarcoplasmic reticulum phospholipids could therefore exert a role on the activity of enzyme proteins which are integral part of the sarcoplasmic reticulum membranes structure, such as the ATPase for Ca*‘or on the membrane permeability transport, and other intrinsic proteins, characteristics [ 311. Alternatively, the presence of the stearyl-CoA desaturase system in the isolated sarcoplasmic reticulum from rabbit slow muscle may reflect the existence of areas of specialization in the intact sarcoplasmic reticulum of this muscle type, as suggested previously by us [9], and could therefore be functionally unrelated to the Ca*‘-transport activity of the sarcoplasmic reticulum membranes. Acknowledgements This work was supported in part by a grant from Muscular Dystrophy Association. The skillful technical assistance of Mr. Renato Siligardi is gratefully acknowledged. We also would like to thank Professor Lars Ernster for helpful advice in the course of this investigation. References 1
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