MITOCHONDRIAL PROTEINS OF FAST-TWITCH GLYCOLYTIC, FAST-TWITCH GLYCOLYTIC-OXIDATIVE AND SLOW-TWITCH-OXIDATIVE RABBIT SKELETAL MUSCLE 0. TAKACS, P. W. D. &LOWSKI*, M. ~YWWO* and F. GUBA Institute of Biochemistry, Szeged University of Medical Sciences, 6701 Szeged. Hungary (Received
16 January
1979)
Abstract-l.
Proteins of mitochondria isolated from rabbit slow-twitch oxidative muscle (m. soleus), fast-twitch-glycolytic muscle (m. psoas) and fast-twitch-glycolytic-oxidative muscle (m. gastrocnemius) were subjected to isoelectrofocusing and subsequent sodium dodecyl sulphate gel electrophoresis. 2. About 120 protein components have been shown to exist in the mitochondria investigated, their molecular weights ranging from 10,000 to 200,000 and isoelectric points from 5.5 to 8.7. 3. Although the general protein pattern of the mitochondria from different muscle types investigated was similar, some differences in the protein composition of mitochondria isolated from functionally different types of muscle have been found.
INTRODUCTION Unlike the protein composition of myofibrils (Perry, 1974; Gauthier & Lowey, 1977) sarcoplasm (Takacs et al., 1977) and sarcoplasmic reticulum (Martonosi, 1972) from fast and slow skeletal muscle, the differences between the mitochondria from different types of muscle has attracted little attention. No data are available on the composition of proteins from isolated skeletal muscle mitochondria. Investigations of liver mitochondrial proteins (Curtis, 1970; Schnaitman, 1969; Melnick et al., 1973) revealed that about 20 components are detectable on gels after sodium dodecyl sulphate electrophoresis, ranging in molecular weight from 10,000 to 140,000 daltons. In the mitochondria from brown fat tissue Ricquier & Kader, (1976) were able to show 17 polypeptide bands with apparent molecular weights of 26,000 to 120,000 daltons. The aim of this paper is to characterize protein composition of different skeletal muscle mitochondria by using a joint technique of electrophoresis and isoelectrofocusing (Laemmli, 1970; O’Farrell, 1975) particularly suitable for comparison of complex protein mixtures.
solution containing 1% SDS, 5% beta-mercaptoethanol and 10% sucrose. Aliquots (2C4O~I) of the solubilized material were put on gel slabs containing 1% SDS. In some experiments a discontinuous polyacrylamide gel gradients (5%, IO’?, 15%) were used. Myofibrillar proteins were run concomitantly as standards. For two dimensional separation by isoelectrofocusing and electrophoresis the mitochondrial pellet was dissolved in a solution containing: 8 M urea, O.lO,:, Nonidet P-40, 50mM dithiothreitol and 2% Ampholine pH 3.5-10, at room temperature to make the protein concentration IOmg per ml. The samples were then dialysed against the same solution for 8 hr and next for further 16 hr against the solution containing the same compounds but 5 mM dithiothreitol. The samples were then focused in glass tubes of 2.5 mm inner diameter as described by (O’Farrell et al., 1977). Following the isoelectrofocusing the gel rods were equitibrated for 30min in the Tris-glycine buffer pH 8.3 containing 0.1% SDS and subjected to electrophoresis in the second dimension as described by (Laemmli, 1970). The gel slabs were stained with Coomassie Blue by the method of (Weber & Osborn, 1969). The stained one dimensional gels were evaluated with KippZonnen densitometer, the two-dimensional maps were photographed. Protein concentration was determined by means of the biuret reaction. Reagents
MATERIALS AND METHODS Mitochondria were isolated as described by ($Hiierczyriski et al., 1975) from three functionally different muscles of normal adult New Zealand white rabbits: soleus as slow-twitch-oxidative muscle, psoas as fast-twitch-glycolytic muscle and gastrocnemius as fast-twitch-glycolytic-oxidative (mixed type) muscle. One dimensional polyacrylamide sodium dodecyl sulphate SDS gel electrophoresis was carried out essentially according to (Tabolt & Yphantis, 1971) after the amount of mitochondria corresponding to 500 pg protein had been solubilized
by heating
for 1 min at 100°C in 100~1 of the
*Present address: Department of Biochemistry. Medical School
in Gdarisk,
SO-21
I Gdalisk,
Poland. 859
Ampholine pH 3.5-10 was a product of LKB (Sweden). Acrylamide, methylenebisacrylamide and N,N,N’,N’-tetramethylenediamine were obtained from Kodak-Eastman Co., (U.S.A.), Coomassie brillant blue R-250 from Mann Chemicals (England). Sodium dodecyl sulphate SDS was purchased from Sigma Chemicals Co. (U.S.A.) and crystallized from 95% ethanol. All other reagents were of highest purity grade obtainable from Reanal (Hungary). RESULTS AND DISCUSSION
While using one dimensional SDS gel electrophoresis, about 40 components could have been obtained from the mitochondria of all types of skeletal muscle investigated. The molecular weights of these components are listed in Table 1. At least six
860
0. TAKACS. P. W. D. SCISLOWSKI, M. ~YDOWO
Table
1. Relative
mobilities
(Rf)
and
and F, GUBA
molecular weights proteins
(mol. wt) of skeletal muscle mitochondria1 Band
no.
1 2 3 4 5 6 7 8 9 10 11 12 I3 14 15 16 17 18 :z
Rf
0.866 0.83 0.778 0.77 0.15 0.72 0.71 0.68 0.67 0.64 0.61 0.59 0.56 0.54 0.53 0.48 0.47 0.45 0.42 0.39
Mol. wt.
Band
x 103
no.
II.0 12.2 14.6 15.0 16.0 17.8 18.2 20.0 21.0 22.9 25.1 27.0 29.5 31.5 33.0 38.5 45.0 48.5 50.0 54.0
:: 23 24 25 26 21 28 :;: 31 32 33 34 35 36 37 38 40 39
Mol. wt
X 103
Rf 0.35 0.3 I 0.29 0.26 0.24 0.22 0.20 0.19 0.16 O.IS 0.14 0.12 0.08 0.06 0.05 0.045 0.034 0.019 0.005 0.014
Bands numbers refer to the gel scanner presented in Fig. I. Myofibrillar proteins
60.0 71.0 74.5 83.0 85.0 94.5 103.0
105.0 114.0 120.0 125.5 132.0 148.0 162.0 167.0 171.0 175.0 185.0 200.0 190.0
tracing
peaks
were used as standards for the molecular weight estimations. which are presented as mean values from three experiments, SE within 10%.
com~nents (marked 2, 3, 6, 7, 12 and 20 on Fig. 1) appeared to be common major proteins of mitochondria from psoas, gastrocnemius and soleus. These components constituted in all the mitochondria species about 30% of the total protein material, as judged from the surface area of the corresponding peaks on gel scanner tracings presented in Fig. I. Marked differences between muscle types were visible in the region of higher molecular weights components. The band No. 32 (molecutar weight 132,000) was abundant in the mitochondria of soleus muscle whereas it was found to be present in a smati proportion in gastr~nemius and absent in psoas. The psoas muscle however contained a much higher proportion of the components No. 30 and 33-38, which were
92 87
8 75 7 6.8 h5
8 I2 60 85 86 89 102 23 70 20 21 119 120
Molecular weight (daltons) 11.000
IO,500 48,000 IO5,OOO 105.000
I os,ooo 150,ooo
26,000 75.000 26,000 26.000 180,000 180,000
Isoelectric point PH 5.6 5.5 7.7 8.5 8.3 6.6 6.7 6.1 7.8 6.9 6.8 6.7 6.6
55
5 Pt-r
Fig. 3. Comparison of the main mitochondrial protein components occurring in the slow. fast and mixed type skeletal muscle. The diagram was obtained by superimposing two-dimensional separation maps from m. soleus. m. psoas and m. gastrocnemius mitochondria: e--common major components, B-slow major components, A-mixed major components, c3-slow minor components, A-mixed minor components. --- --distribution pattern of minor components enclosed by lines.
less abundant in both gastr~nemius and soieus. Atso the band No. 22 seems to be present in larger amount in the mit~bondria of slow psoas muscle. By using two-dimensional separation technique, about 120 protein components have been shown to be present in skeletal muscle mitochondria. An example of a separation of proteins from muscle mitochondria is presented in Fig. 2. Figure 3 presents a diagram comparing the components in the mitochondria of all the three types of muscle. As may be seen from Table 2 at least three protein components were present in soleus muscle only, three others were present in soleus and gastrocnemius but absent in the mitochondria from psoas muscle. Isoelectric points of the proteins separated by the two dimensional technique ranged from pH 5.5 to 8.7, their molecular weights from lO,OOOto 180,~.
Table 2. Some major protein components of rabbit skeletal muscle mitochondria spot no.
6
Psoas
Soleus
Gastrocnemius
+ t + + + + +
+ + + + + + +
f + + + + + +
A.. -
+ + + + +
_ _ + + +
+
-
Solubilized mitochondrial proteins were subjected to isoelectrofocusing and SDS electrophoresis as described in the text. The molecular weight values within f IOU/,.
M. SOLEUS
MITOCHONDRIAL
PROTEINS
Fig. l(a).
M. PSOAS
MITOCHONDRIAL
‘8
B
Fig. l(b).
861
PROTEiNS
M. GASTROCNEMIUS
MITOCHONDRIAL
PROTEINS
Fig. l(c)
Fig. 1. Separation of ~~~~c~~n~~i~~ proteins by SDS-gel efectruphoresis. a---miwchondria isolated from m. s3ieu.s. b-from m. psoas, c-from m. gastrocnemius of adult rabbits of the white Ne& Zealand strain. For experimental details see text.
Fig. 2. Two-dimensional separation of mitochondrial proteins from rabbit soleus muscle. Isoelectrofocusing (IF) was carried out in the first direction and sodium dodecyl sulphate electrophoresis (SDS) in the second direction. For experimental details see the text.
Fast and slow muscle mitochondria
The number of components extracted from skeletal muscle mitochondria by our procedure is comparable to the number of protein bands found by Richter et al. (1978) in heart muscle mitochondria. These authors investigated the extractability of proteins from heart mitochondria by volatile anesthetics and showed about 40 bands while subjecting solubilized whole mitochondria to SDS electrophoresis. The diversity of the protein composition of the mit~hondria from slow, fast and mixed type of skeletal muscle shown in this paper is correlated probably in some way with the metabolic differences of these organelles in different muscle types. Although some differences between the metabolic functions of mitochondria from red and white skeletal muscle have been found in the past (Rigault & Blanchaer, 1970) much work has to be done before it will be possible to put the functional differences in molecular terms.
REFERENCES CURTIS
proteins
865
ation and characterization by polyacrylamide gel electrophoresis. Biochim. hiophys. Acta 311, 23G241. G’FARRELLP. H. (1975) High resolution two-dimensional electrophoresis of protein. J. biol. them. 250, 4007-402 I. O’FARRELLP. Z., GOODMANH. M. & O’FARRELLP. H. (1977) High-resolution two-dimensional electrophoresis of basic as well as acidic proteins. Celi 12, 1133-I 142. PERRY S. V. (1974) In Euplorarory Concepts if? Muscular Dystrophy. (Edited by MILHORATA. T.). 2nd edn., pp. 3 19-328. Excerpta Medica, Amsterdam. RICQUIERD. & KADERJ. C. (1976) Mitochondrial protein alternation in active brown fat.: A sodium dodecyl sulphate poiyacrylamide gel electrophoretic study. Biochem. biophys.
Res. Commun.
73, 577-583.
RICHTERJ. .I, SUNI~ERLAND E.. JUHL U. & KORNGHUTS. (1978) Extraction on mitochondrial proteins by volatile anesthetics. Biochim. hiophys. Acta 543, 106-I 15. RIGAULTM. Y. A. & BLANCHAER M. C. (1970) Respiration and oxidative phosphorylation by mitochondria of red and white skeletal muscle. Gun. J. Biochem. 48, 27-32. SCHNAITMAN C. A. (1969) Comparison of rat liver mitochondrial and microsomal membrane proteins. Biochemistry
63, 4 12-4 19.
SWIERCZY~~SKI J., ALEKSAN~ROWICZ 2. & ZYWWO M. (1975) Effect of some steroids and a-tocopherol on cytochrome c induced extramitochondrial NADH oxidation by human and rat skeletal muscle mitochondria. Itit. J.
P. J. (1970) The molecular weight of the major protein component from mito~hondriai membranes. Biochim. biop~ys. Actu 211, 57-577. Biochem. 6, 757-763. GAUTHKR G. F. & L~WEYS. (1977) Polymorphism of myosin among skeletal muscle fiber types. J. Celi Biol. 74, TABOLTD. N. & YPHANTISD. A. (1971) Fluorescent monitoring of SDS-gel electrophoresis. Anaiyr. Biochem. 44, 760-779. 246254. LAEMMLIU. K. (1970) Cleavage of structural proteins durTAKACS0.. SOHARI., SZILAGYI T. & GUBA F. (1977) Exing assembly of the head of bacteriophage T4. Narure perimental investigations on hypokinesis of skeletal 227, 680-685. muscles with different function. IV. Acra biol. Sci. Hung. MARTONOS~ A. (1972) In Current Topics in Membranes and 28, 221-230. Trunsport (Edited by BONNERF. & KLEINZELLERA.). Vol. 3, pp. 83-197. Academic Press, New York. WEBERK. & OSBORNM. (1969) The reliability of molecular MELNICKR. L., TINBERGH. M., MAGUIREJ. & PACKER weight determinations by dodecyl sulphate polyacrylamide gel electrophoresis. J. hiot. them. 244, 44064412. L. (1973) Studies on mitochondrial proteins. I. Separ-
16 January
1979)
Abstract-l.
Proteins of mitochondria isolated from rabbit slow-twitch oxidative muscle (m. soleus), fast-twitch-glycolytic muscle (m. psoas) and fast-twitch-glycolytic-oxidative muscle (m. gastrocnemius) were subjected to isoelectrofocusing and subsequent sodium dodecyl sulphate gel electrophoresis. 2. About 120 protein components have been shown to exist in the mitochondria investigated, their molecular weights ranging from 10,000 to 200,000 and isoelectric points from 5.5 to 8.7. 3. Although the general protein pattern of the mitochondria from different muscle types investigated was similar, some differences in the protein composition of mitochondria isolated from functionally different types of muscle have been found.
INTRODUCTION Unlike the protein composition of myofibrils (Perry, 1974; Gauthier & Lowey, 1977) sarcoplasm (Takacs et al., 1977) and sarcoplasmic reticulum (Martonosi, 1972) from fast and slow skeletal muscle, the differences between the mitochondria from different types of muscle has attracted little attention. No data are available on the composition of proteins from isolated skeletal muscle mitochondria. Investigations of liver mitochondrial proteins (Curtis, 1970; Schnaitman, 1969; Melnick et al., 1973) revealed that about 20 components are detectable on gels after sodium dodecyl sulphate electrophoresis, ranging in molecular weight from 10,000 to 140,000 daltons. In the mitochondria from brown fat tissue Ricquier & Kader, (1976) were able to show 17 polypeptide bands with apparent molecular weights of 26,000 to 120,000 daltons. The aim of this paper is to characterize protein composition of different skeletal muscle mitochondria by using a joint technique of electrophoresis and isoelectrofocusing (Laemmli, 1970; O’Farrell, 1975) particularly suitable for comparison of complex protein mixtures.
solution containing 1% SDS, 5% beta-mercaptoethanol and 10% sucrose. Aliquots (2C4O~I) of the solubilized material were put on gel slabs containing 1% SDS. In some experiments a discontinuous polyacrylamide gel gradients (5%, IO’?, 15%) were used. Myofibrillar proteins were run concomitantly as standards. For two dimensional separation by isoelectrofocusing and electrophoresis the mitochondrial pellet was dissolved in a solution containing: 8 M urea, O.lO,:, Nonidet P-40, 50mM dithiothreitol and 2% Ampholine pH 3.5-10, at room temperature to make the protein concentration IOmg per ml. The samples were then dialysed against the same solution for 8 hr and next for further 16 hr against the solution containing the same compounds but 5 mM dithiothreitol. The samples were then focused in glass tubes of 2.5 mm inner diameter as described by (O’Farrell et al., 1977). Following the isoelectrofocusing the gel rods were equitibrated for 30min in the Tris-glycine buffer pH 8.3 containing 0.1% SDS and subjected to electrophoresis in the second dimension as described by (Laemmli, 1970). The gel slabs were stained with Coomassie Blue by the method of (Weber & Osborn, 1969). The stained one dimensional gels were evaluated with KippZonnen densitometer, the two-dimensional maps were photographed. Protein concentration was determined by means of the biuret reaction. Reagents
MATERIALS AND METHODS Mitochondria were isolated as described by ($Hiierczyriski et al., 1975) from three functionally different muscles of normal adult New Zealand white rabbits: soleus as slow-twitch-oxidative muscle, psoas as fast-twitch-glycolytic muscle and gastrocnemius as fast-twitch-glycolytic-oxidative (mixed type) muscle. One dimensional polyacrylamide sodium dodecyl sulphate SDS gel electrophoresis was carried out essentially according to (Tabolt & Yphantis, 1971) after the amount of mitochondria corresponding to 500 pg protein had been solubilized
by heating
for 1 min at 100°C in 100~1 of the
*Present address: Department of Biochemistry. Medical School
in Gdarisk,
SO-21
I Gdalisk,
Poland. 859
Ampholine pH 3.5-10 was a product of LKB (Sweden). Acrylamide, methylenebisacrylamide and N,N,N’,N’-tetramethylenediamine were obtained from Kodak-Eastman Co., (U.S.A.), Coomassie brillant blue R-250 from Mann Chemicals (England). Sodium dodecyl sulphate SDS was purchased from Sigma Chemicals Co. (U.S.A.) and crystallized from 95% ethanol. All other reagents were of highest purity grade obtainable from Reanal (Hungary). RESULTS AND DISCUSSION
While using one dimensional SDS gel electrophoresis, about 40 components could have been obtained from the mitochondria of all types of skeletal muscle investigated. The molecular weights of these components are listed in Table 1. At least six
860
0. TAKACS. P. W. D. SCISLOWSKI, M. ~YDOWO
Table
1. Relative
mobilities
(Rf)
and
and F, GUBA
molecular weights proteins
(mol. wt) of skeletal muscle mitochondria1 Band
no.
1 2 3 4 5 6 7 8 9 10 11 12 I3 14 15 16 17 18 :z
Rf
0.866 0.83 0.778 0.77 0.15 0.72 0.71 0.68 0.67 0.64 0.61 0.59 0.56 0.54 0.53 0.48 0.47 0.45 0.42 0.39
Mol. wt.
Band
x 103
no.
II.0 12.2 14.6 15.0 16.0 17.8 18.2 20.0 21.0 22.9 25.1 27.0 29.5 31.5 33.0 38.5 45.0 48.5 50.0 54.0
:: 23 24 25 26 21 28 :;: 31 32 33 34 35 36 37 38 40 39
Mol. wt
X 103
Rf 0.35 0.3 I 0.29 0.26 0.24 0.22 0.20 0.19 0.16 O.IS 0.14 0.12 0.08 0.06 0.05 0.045 0.034 0.019 0.005 0.014
Bands numbers refer to the gel scanner presented in Fig. I. Myofibrillar proteins
60.0 71.0 74.5 83.0 85.0 94.5 103.0
105.0 114.0 120.0 125.5 132.0 148.0 162.0 167.0 171.0 175.0 185.0 200.0 190.0
tracing
peaks
were used as standards for the molecular weight estimations. which are presented as mean values from three experiments, SE within 10%.
com~nents (marked 2, 3, 6, 7, 12 and 20 on Fig. 1) appeared to be common major proteins of mitochondria from psoas, gastrocnemius and soleus. These components constituted in all the mitochondria species about 30% of the total protein material, as judged from the surface area of the corresponding peaks on gel scanner tracings presented in Fig. I. Marked differences between muscle types were visible in the region of higher molecular weights components. The band No. 32 (molecutar weight 132,000) was abundant in the mitochondria of soleus muscle whereas it was found to be present in a smati proportion in gastr~nemius and absent in psoas. The psoas muscle however contained a much higher proportion of the components No. 30 and 33-38, which were
92 87
8 75 7 6.8 h5
8 I2 60 85 86 89 102 23 70 20 21 119 120
Molecular weight (daltons) 11.000
IO,500 48,000 IO5,OOO 105.000
I os,ooo 150,ooo
26,000 75.000 26,000 26.000 180,000 180,000
Isoelectric point PH 5.6 5.5 7.7 8.5 8.3 6.6 6.7 6.1 7.8 6.9 6.8 6.7 6.6
55
5 Pt-r
Fig. 3. Comparison of the main mitochondrial protein components occurring in the slow. fast and mixed type skeletal muscle. The diagram was obtained by superimposing two-dimensional separation maps from m. soleus. m. psoas and m. gastrocnemius mitochondria: e--common major components, B-slow major components, A-mixed major components, c3-slow minor components, A-mixed minor components. --- --distribution pattern of minor components enclosed by lines.
less abundant in both gastr~nemius and soieus. Atso the band No. 22 seems to be present in larger amount in the mit~bondria of slow psoas muscle. By using two-dimensional separation technique, about 120 protein components have been shown to be present in skeletal muscle mitochondria. An example of a separation of proteins from muscle mitochondria is presented in Fig. 2. Figure 3 presents a diagram comparing the components in the mitochondria of all the three types of muscle. As may be seen from Table 2 at least three protein components were present in soleus muscle only, three others were present in soleus and gastrocnemius but absent in the mitochondria from psoas muscle. Isoelectric points of the proteins separated by the two dimensional technique ranged from pH 5.5 to 8.7, their molecular weights from lO,OOOto 180,~.
Table 2. Some major protein components of rabbit skeletal muscle mitochondria spot no.
6
Psoas
Soleus
Gastrocnemius
+ t + + + + +
+ + + + + + +
f + + + + + +
A.. -
+ + + + +
_ _ + + +
+
-
Solubilized mitochondrial proteins were subjected to isoelectrofocusing and SDS electrophoresis as described in the text. The molecular weight values within f IOU/,.
M. SOLEUS
MITOCHONDRIAL
PROTEINS
Fig. l(a).
M. PSOAS
MITOCHONDRIAL
‘8
B
Fig. l(b).
861
PROTEiNS
M. GASTROCNEMIUS
MITOCHONDRIAL
PROTEINS
Fig. l(c)
Fig. 1. Separation of ~~~~c~~n~~i~~ proteins by SDS-gel efectruphoresis. a---miwchondria isolated from m. s3ieu.s. b-from m. psoas, c-from m. gastrocnemius of adult rabbits of the white Ne& Zealand strain. For experimental details see text.
Fig. 2. Two-dimensional separation of mitochondrial proteins from rabbit soleus muscle. Isoelectrofocusing (IF) was carried out in the first direction and sodium dodecyl sulphate electrophoresis (SDS) in the second direction. For experimental details see the text.
Fast and slow muscle mitochondria
The number of components extracted from skeletal muscle mitochondria by our procedure is comparable to the number of protein bands found by Richter et al. (1978) in heart muscle mitochondria. These authors investigated the extractability of proteins from heart mitochondria by volatile anesthetics and showed about 40 bands while subjecting solubilized whole mitochondria to SDS electrophoresis. The diversity of the protein composition of the mit~hondria from slow, fast and mixed type of skeletal muscle shown in this paper is correlated probably in some way with the metabolic differences of these organelles in different muscle types. Although some differences between the metabolic functions of mitochondria from red and white skeletal muscle have been found in the past (Rigault & Blanchaer, 1970) much work has to be done before it will be possible to put the functional differences in molecular terms.
REFERENCES CURTIS
proteins
865
ation and characterization by polyacrylamide gel electrophoresis. Biochim. hiophys. Acta 311, 23G241. G’FARRELLP. H. (1975) High resolution two-dimensional electrophoresis of protein. J. biol. them. 250, 4007-402 I. O’FARRELLP. Z., GOODMANH. M. & O’FARRELLP. H. (1977) High-resolution two-dimensional electrophoresis of basic as well as acidic proteins. Celi 12, 1133-I 142. PERRY S. V. (1974) In Euplorarory Concepts if? Muscular Dystrophy. (Edited by MILHORATA. T.). 2nd edn., pp. 3 19-328. Excerpta Medica, Amsterdam. RICQUIERD. & KADERJ. C. (1976) Mitochondrial protein alternation in active brown fat.: A sodium dodecyl sulphate poiyacrylamide gel electrophoretic study. Biochem. biophys.
Res. Commun.
73, 577-583.
RICHTERJ. .I, SUNI~ERLAND E.. JUHL U. & KORNGHUTS. (1978) Extraction on mitochondrial proteins by volatile anesthetics. Biochim. hiophys. Acta 543, 106-I 15. RIGAULTM. Y. A. & BLANCHAER M. C. (1970) Respiration and oxidative phosphorylation by mitochondria of red and white skeletal muscle. Gun. J. Biochem. 48, 27-32. SCHNAITMAN C. A. (1969) Comparison of rat liver mitochondrial and microsomal membrane proteins. Biochemistry
63, 4 12-4 19.
SWIERCZY~~SKI J., ALEKSAN~ROWICZ 2. & ZYWWO M. (1975) Effect of some steroids and a-tocopherol on cytochrome c induced extramitochondrial NADH oxidation by human and rat skeletal muscle mitochondria. Itit. J.
P. J. (1970) The molecular weight of the major protein component from mito~hondriai membranes. Biochim. biop~ys. Actu 211, 57-577. Biochem. 6, 757-763. GAUTHKR G. F. & L~WEYS. (1977) Polymorphism of myosin among skeletal muscle fiber types. J. Celi Biol. 74, TABOLTD. N. & YPHANTISD. A. (1971) Fluorescent monitoring of SDS-gel electrophoresis. Anaiyr. Biochem. 44, 760-779. 246254. LAEMMLIU. K. (1970) Cleavage of structural proteins durTAKACS0.. SOHARI., SZILAGYI T. & GUBA F. (1977) Exing assembly of the head of bacteriophage T4. Narure perimental investigations on hypokinesis of skeletal 227, 680-685. muscles with different function. IV. Acra biol. Sci. Hung. MARTONOS~ A. (1972) In Current Topics in Membranes and 28, 221-230. Trunsport (Edited by BONNERF. & KLEINZELLERA.). Vol. 3, pp. 83-197. Academic Press, New York. WEBERK. & OSBORNM. (1969) The reliability of molecular MELNICKR. L., TINBERGH. M., MAGUIREJ. & PACKER weight determinations by dodecyl sulphate polyacrylamide gel electrophoresis. J. hiot. them. 244, 44064412. L. (1973) Studies on mitochondrial proteins. I. Separ-
