0020.71 IX;8107Og43-07SO2.~/0 Q 1981 Pergamon Press Ltd
DISSIMILARITY OF CYTOSOL AND MITOCHONDRIAL MALIC ENZYME FROM RAT SKELETAL MUSCLE JULIAN SWIERCZY~SKI Department of Biochemistry, I.B.M. Medical School. 8&211 Gdarisk, ul. Dgbinki 1, Poland (Rewired
Abstract-l, The distribution and some properties of malic enzyme (L-malate-NADP’ oxidoreductase (oxaloacetate-decarboxylating), EC 184.108.40.206) in rat hindlimb skeletal muscle have been investigated. 2. Abaut 30”); of the total activity is present intramitochondrially. 3. The cytosol and mitochondrial malic enzymes were shown to be different in their chromatographic behaviour on DEAE-cellulose. electrophoretic mobilities and the extent of inhibition by DTNB. dicoumarol and acetyl-CoA. 4. The isoelectric point for mitochondrial and cytoptasmic enzyme was 6.55 and 6.15 respectively. 5. The molecular weights of the two isozymes were similar as determined by gel filtration.
mitochondrial malic enzymes from porcine heart with regard to inhibition by acetyl-CoA. The present study was undertaken to obtain more information on differences between mitochondrial and cytoplasmic malic enzyme from rat skeletal muscle.
It is well established that two different types of ~ADP-linked malic enzymes are present in most mammalian tissues (Henderson, 1966; Simpson & Estabrook, 1969; Frenkel, 1971. 1972; Bartholome et (I/.. 1972; Brdiczka & Pette, 1972). One of these enzymes is located in the cytosol whereas the other is present in the mitochondrial matrix. The experiments of Nolte et ul. (1972) indicated that in the case of skeletal muscle the malic enzyme activity is also
bimodally distributed in cytosol and mitochondira. In the red muscle about 70”, of the total activity is located in the mitochondria, whereas in white muscle up to 70”, is found,in the cytosol (Nolte et al., 1972). In recent papers (Swierczynski et al., 1980; Swierczynski, 1980; Swierczynski, in press) we have reported purification and some kinetic properties of the mitochondrial and cytoplasmic malic enzyme from rat skeletal muscle. We have shown that the isozymes differ in their kinetic properties, since only the mitochondrial enzyme showed cooperativity at low concentration of malate. Another striking difference between the two enzymes was the ratio of the abilities to catalyze the decarboxylation and carboxylation reaction, The reaction catalyzed by the mitochondrial
malic enzyme showed negligible reversibility, but extramitochondrial malic enzyme appeared to catalyze the reaction readily in both directions. It seems therefore unlikely, that malic enzyme present skeletal muscle cytosol is the mitochondrial
malic enzyme which leaked out into the cytosol during the cell fractionation. Nevertheless no proof has been presented so far that those are in fact two molecular species. It has been shown that the mitochondrial malic enzyme of heart (Frenkel, 1971), adrenal cortex (Simpson & Estabrook, 1969) and brain (Frenkel, 1972:
Frenkel & Cobo-Frenkel, 1973) is different from that of the cytosol in several respects such as electrophoretic or chromatographic behaviour on DEAEcellulose and the extent of inhibition by dicoumarol or by sulfhydryl reagents. Bartholome et al. (1972) also reported differences between the cytoplasmic and *c
The following chemicals were purchased from Sigma Chemical Co. (U.S.A.): NADP. malic acid. phenazine methosulphate, nitro blue tetrazolium, DTNB (5SDithiobis-(2-nitrobenzoic acid)) and acetyl-CoA. Sepharose 6B and ADP-sepharose were from Pharmacia Fine Chemicals, Ultrogel AcA-34 and Ampholine were from LKB (Sweden). DEAE-cellulose was from Whatman Biochemicals Ltd. Di~oumarol (3.3 methylenbis(4-hydroxycumarin)) was purchased from Merck Schuchardt. All other chemicals were of the highest purity available from POCH (Gliwice, Poland). Male Wistar rats maintained on a commercial complete diet were used for experiments. Rat skeletal muscle obtained from the hind legs immediately after decapitation was freed of connective and adipose tissue, minced finely with scissors and rinsed thoroughly with isotonic K-Cl. Mitochondria were prepared as described previously (Swierczynski rt ul., 1975). Postmitochondrial supernatant was additionally centrifuged at 20.000 g for 30 min and used as a source of extramitochondriai malic enzyme. The mitochondrial and cytosol malic enzymes from rat skeletal muscle were prepared as described previously (Swierczynski et ccl.,1980; Swierczynski, 1980). Isoelectric focusing was carried out in sucrose density gradient according to the LKB manual with a narrow range of ampholine pH S-7, in LKB model 8100 apparatus The enzyme was dialyzed against lo/, glycine before application to the column. Electrofocusing was performed at 600 V and 4 C. continued for 24 hr and the pH of the 2.5 ml fractions was measured immediately after elution at 4’C. Folyacrylamide, gel electrophoresis was performed as described recently (Swierczynski, 1980).
The enzyme activity was followed spectrophotometritally with Specord U.V.Vis recording spectrophotometet by observing the appearance of NADPH at 340 nm and 30-C.
for malic enzyme assay 50mM Tr+HCI pH 7.2, IOmM r-malate. 1 mM MnC12. 0.5 mM NADP and the enzyme in amounts which caused the increase of absorbance at the range 0.1-0.2 min. Protem concentrations uere determined by the method of Spector (197X). The standard
(tinal volume I ml) contained:
Postmitochondrial supernatant which had been additionally centrifuged at 20,000 g for 30 min was used .for purification of cytoplasmic malic enzyme (Swierczytiski. 1980). Muscle homogenization and a rather long lasting procedure of isolating mitochondria by di~erential centrifugation may damage the mitochondria causing that soluble mitochondriai malic enzyme appearing in the cytoplasmic fraction. The results given in Table 1 show that about 30”. of malic enzyme was retained in mitochondria isolated from mixed types of rat skeletal muscle. This indicates that intracellular distribution of malic enzyme in mixed types of skeletal muscle obtained by our procedure is similar to that described by Nolte c’r trl. (1972). These authors were using an indirect. but relatively milder technique of fractionati~)n of muscle. in which the tissue was gently disrupted and stirred in a nearisoosmotic medium: by this procedure the soluble enzymes of the cytoplasm are released into the medium whereas mitochondrial enzymes are retained in the tissue. It should be pointed out that total activity extracted in our experiments was also vzery similar to that obtained by Newsholmc & Williams (1978). Thus our procedure appears to be satisfactory to study intracellular localization as there is no indtcation that any serious loss of the enzyme activity during isolation procedure did occur. The results presented in Table 1 also indicate that in the case of rat sekeletal muscle the malic enzyme is distributed both in mitochondria and in the cytoplasmic fraction, To check whether the mitochondrial and cytoplasmic malic enzymes represent two different forms of malic enzyme. the cnzymcs isolated from both fractions Table I. Intr;lccllular
distribution m l-at skeletal
were rechromatographed on DEAE-cellulose under identical conditions. It is well established that the mito~hondrial and cytosol variants from bovine(Frenkel. 1971) rat- (Isohashi et al.. 1971) and pighearts (Bartholome ef trl.. 1972) as well as bovine brain (Frenkel. 1972; Frenkel & Cobo-Frenkel. 1973) and adrenal cortex (Simpson & Estabrook, 1969) show clear differences in their cllromatographic mobiIities. Cytoplasmic malic enzyme from bovine and adrenal cortex could be eluted from DEAE-cellulose column vvith 10 mM MgClz whereas mitochondrial malic enzyme required higher concentration of MgClz [40 mM). Berstine (1979) obtained also a good resolution of mitochondrial and cytoplasmic mahc enzyme on DEAE-cellulose column with linear gradient of KCI. In the experiment presented in Fig. 1 the cytosol and mitochondrial malic enzyme of rat skeletal muscle was applied on a column (2.5 x 45cm) of
r .i !i I i
i i, i l l
t . . ..-0.W.’
I i : t ,
of malic enzyme activit! muscle
28.6 71 2 8.5
Skeletal muscle from the hind legs of rat ~3s freed of connectlbe and adipose tissue. minced tincly bvith scissors and rinsed thoroughly with isotontc KCI The muscle lcilS the suspended in approx. IO ~ol of cold medium containin%: 0.71 M manitoi. 0.07 M sucrose. 0.01 M EDNA and 0.61 M Trjs--HCI rpH 7.8) and \uas mixed for 30sec in an omnimixer. The resulting homogenate M’S centrifuged 5 min at @fJ 9, Supernatant (designated as homogenate) was decanted and centrifuged for 15 mm at 14.OOOg. The supcrnatant obtamed was da&mated as cytoplasmic fraction. The peilct HIS suspended in the isolatron medium and designated ~$5mitochondrl~l fraction. Enzyme activity ws assayed as described in the text at 30 C in the medium containing: 50mM Tris-WC1 pH 7.5. O.l”,, Triton X-100. 0.5 mM NADP. 2 mM MnCl: and 10 mM L-malate.
Enqme activity (~tmoi min per 100 g fresh vkght of muscle) Homogenate Cytoplasmic fraction ~lit~~chondri~li fraction
t _ : 30
Tube number Fig. 1. DEAE-cellulose chromatography of mahc enLyme. Cytoplasmic (A). mitochondrial IB) and the mixture of both isozymes (C) were loaded on DEAE-cellulose column and eluted with linear gradient of KCI. Fractions (4.5 ml) were collected and assayed for malic enzyme activity as described under Materials and Methods. Other conditions see text.
DEAE-cellulose preequilibrated with 10 mM TrisHCI (pH 7.8) plus 2 mM EDTA (pH 7.8). The column was eluted with a linear gradient generated by 250 ml each of the same buffer and of the buffer containing 0.3 M KCI. Under these conditions, the activity of mitochondrial (Fig. 1B) and cytoplasmic (Fig. 1A) malic enzyme came off in a single symmetrical peak. When the mitochondrial and cytoplasmic enzymes were mixed and then chromatographed on DEAEcellulose column under the same conditions only a partial separation of the enzymes was achieved (FIN. 1C). These results seem to indicate a significant difference in chromatographic behaviour on DEAEcellulose between the cytosol and mitochondrial malic enzymes from rat skeletal muscle. Hence one may suppose that cytoplasmic and mitochondrial malic enzymes from rat skeletal muscle are different proteins. This was proved also by electrofocusing experiment presented in Fig. 2. The cytoplasmic and mito06
T IO6. 64-
Fig. 2. Isoelectric focusing of cytoplasmic malic enzyme (A). mitochondrial malic enzyme (B) and mixture of both lsozymes (C). Experimental conditions as described under Materials and Methods. In experiment (C) malic enzyme was assayed in the absence (M) or in the presence (after IO min preincubation) of 20 pm DTNB ( x ~ x ).
chondrial malic enzymes were subjected to isoelectric focusing on a pH 5-7 ampholine gradient. A single, symmetrical activity peak of cytoplasmic with pI 6.15 (Fig. 2A) and of mitochondrial enzyme with pI 6.55 (Fig. 2B) was obtained. The focusing of a mixture of the mitochundrial and cytoplasmic enzyme resulted in the detection of two peaks with p1 6.15 and 6.55 respectively (Fig. 2C). When the activity was measured in the presence of DTNB an inhibitor of mitochondrial malic enzyme (see Fig. 6) only one peak of activity with pI 6.15 was obtained. Thus the enzyme with p1 6.15 is identical with cytosol malic enzyme and the activity with p1 6.55 with mitochondrial enzyme. It should be pointed out that isoelectric point of cytoplasmic malic enzyme from rat skeletal muscle found in our experiments is different than the isoelectric point of the malic enzyme from the pig-heart cytoplasm (Bartholome rr ul., 1972). The isoelectric point of mitochondrial malic was also different than the isoelectric point of the mitochondrial malic enzyme from pig heart (Bartholome er ul., 1972). although this difference is much smaller. Electrophoretic analysis in polyacrylamide gel of the malic enzymes from rat skeletal muscle is shown in Fig. 3. As may be seen the cytoplasmic and mitochondrial malic enzymes migrated as distinct single bands towards the anode, the slowly migrating isozyme being the isozyme obtained from cytoplasmic fraction. This characteristic is in agreement with the fact that a higher ionic strength is required for the elution of mitochondrial malic enzyme from DEAE-cellulose than for the elution of cytoplasmic malic enzyme. These results are also in good agreement with the different isoelectric points described above. Although mitochondrial and cytoplasmic malic enzymes from rat skeletal muscle could be separated by electrophoresis and ion-exchange chromatography. these enzymes came off a column of ultrogel AcA-34 as a single symmetrical peak. As illustrated in Fig. 4 this peak contained both the DTNB-sensitive and DTNBinsensitive activities of malic enzyme, It appears therefore that the two isozymes have a similar molecular weight. Further differences between cytoplasmic and mitochondrial malic enzyme from rat skeletal muscle refer to the influence of temperature on the catalytic activity. The activities of the mitochondrial and cytoplasmic malic enzymes were studied under substrate saturation conditions in the range of temperatures [email protected]
C. The values obtained are presented in the form of Arrhenius plot in Fig. 5. Discontinuities were observed both in the case of mitochondrial and cytoplasmic malic enzyme, however the transition temperatures were 23 and 27’C respectively. As can be seen in Fig. 5 the energy of activation for the mitochondrial malic enzyme was higher as compared with cytoplasmic malic enzyme. The mitochondrial malic enzyme revealed values of activation energies of 6.0, 12.9 kcaljmol and the cytoplasmic enzyme 3.2, 7.9 kcal/mol. Similar values have been reported by Brandon & Boekel-Mol (1973) and Asami et ul. (1979) for the reaction catalyzed by malic enzyme isolated from plants, however these authors did not observe the discontinuity in the Arrhenius plot. The existence of the discontinuity in the Arrhenius plot of the malic enzymes from rat skeletal muscle suggests that either
L \.\ l\ 2
CDTNBI, pM Tube
Fig. 4. Lltrogel AcA-34 chromatography of the mixture of mitochondrial and cytoplasmic malic enzyme. Mixture of mitochondrial and cytoplasmic malic enzyme was loaded on AcA-34 ultrogel column (2 x 75cm) and eluted with IOmM Tris HCI plus 2 mM EDTA (pH 7.8). Fractions (4.4 ml) were collected and assayed for malic cnzyme activity tn the absence of DTNB (0 ~~~O) and rn the presence (after IOmin preincubation) of 20/1M DTNB (m 01. The arrows indicate elution positron of the blue n) dextran (3 ~~ 0) and hemoglobin (m
the enzymesubstrate complex formation is taking place at two different catalytic sites or that a temperature dependent change in the quaternary structure of the enzyme does occur causing the conformation change of the same active site. The experiments reported by Frenkel (1973) clearly demonstrate a large difference in reactivity of mito-
104/T, K-’ Fig. 5. Arrhenius plots for mitochondrial (a ---o) and cytoplasmic (O----O) malic enzyme from rat skeletal muscle. Enzyme assay was carried out in the medmm containing: 50 mM Tris-HCI pH 7.4. 1 mM MnC12. 0.5 mM NADP and 1OmM t-malate. V his expressed in ktmol x
Frg. 6. Inhibition of rat skeletal muscle malic enzyme by DTNB. The activity of either the cytosol (0 mmmo)or the mitochondrial (G-O) malic enzymes were assayed after IOmin incubation at 15 C in 1OOmM Tris-HCI pH 7.2 in the presence of the indicated final concentrations of DTNB. Reacttons were started by the addition of IOmM L-malatc. I mM MnCl? and 0.5 mM NADP and were carried out at 30 C.
and cytoplasmic malic enzyme from bovine brain towards sulfhydryl reagents. It was therefore interesting to check the effect of sulfhydryl reagents on malic enzymes from rat skeletal muscle. Figure 6 shows the results obtained when isozymes were tested for their sensitivity to DTNB. As shown in the figure. the mitochondrial malic enzyme was strongly inhibited by concentrations of DTNB as low as IO PM. while the cytoplasmic isozyme retained complete activity under identical conditions. The striking difference shown by the cytosol and mitochondrial malic enzymes in their sensitivity towards dicoumarol has been shown to be a clear distinguishing property between the isozymes present in bovine adrenal cortex (Simpson & Estabrook. 1969). A study of the possible effect of dicoumarol on rat skeletal muscle isozymes was therefore undertaken. Figure 7 shows the results of an experiment designated to test this property. As shown in Fig. 7 the mitochondrial enzyme was strongly inhibited by concentrations of dicoumarol as low as 40 PM. while the cytosol isozyme was only slightly inhibited. The finding that DTNB (or dicoumarol) is a strong inhibitor of mitochondrial malic enzyme but does not affect the cytosol enzyme (under the same conditions) in skeletal muscle might be of value in studying individual reaction in the muscle extract. It means that DTNB might be a useful tool for quantitative estimation of cytoplasmic and mitochondrial malic enzyme without fractionation of the tissue. Spydevold rr trl. (1976) have shown that acetylcarnitine reduced pyruvate from malate formation by rat skeletal muscle mitochondria. They suggested that the inhibition of malic enzyme by acetyl-CoA formed from acetyl-carnitine is responsible for inhibition of pyruvate formation. Because of that it was of interest to demonstrate the effect of acetyl-CoA on mitochondrial and cytoplasmic malic enzyme from rat skeletal muscle. As can be seen in Fig. 8 about SO”,, inhibition
Fig. 3. Polyacrylamide gel electrophoresis of native mitochondrial malic enzyme (Ml. cytoplasmic mallc enzyme (C1 and the mixture of both isozymes (M + C). All gels were stained for malic enzyme activity. For experimental conditions see Materials and Methods.
20 IO CDi~rn~~ll , PM
Fig. 7. Inhibition of rat skeletal muscle malic enzyme by dicoumarol. Reactions were carried out at 30 C in the medium containing 100mM Tris-HCl pH 7.2. 1 mM M&I,. 0.4mM NADP. 0.5 mM r-malate and dicoumarol at indicated concentration. Mitochondrialic malic enzyme (00); Cytoplasmic malic enzyme (O----O).
of mitochondriai malic enzyme can be achieved at 1 mM acetyl-CoA. The cytosol enzyme was also inhibited by acetyl-CoA. but to a lesser extent. These resutts are similar to that reported by Bartholome et ul. (1972) who have demonstrated a direct effect of acetyl-CoA on the NADP-linked malic enzyme in pig-heart mitochondria. In contrast Spydevold er ul. (1976) were not able to demonstrate any inhibition with acetyl-CoA levels up to 4.0mM of the NADlinked malic enzyme isolated from heart. Nevertheless, our results support the suggestion of Spydevold ef ul. (1976) that the effect of acetyl-CoA on pyruvate from malate formation by rat skeletal muscle mitochondria could be explained by a direct inhibition of the NADP-linked malic enzyme by acetyl-CoA. Although the physiological meaning of the inhibition of mitochondrial malic enzyme by acetyl-CoA
CAcetyl -CoAl,mM Fig. 8. Inhibition by acetyl-CoA of mitochondrial O-_-O) and cytoplasmic (cam 0) mahc enzyme from rat skeletal muscle. Reactions were carried out at 30 C in the medium containing: 1OOmM Tris-HCI pH 7.2. 1 mM MnCiz. 0.5 mM NADP. 0.2 mM L-malate and acetyl-CoA at indicated final concentrations.
one can argue that it might be important under physiological conditions. Such inhibition could offer a new explanation for the reduction of alanine release from skeletal muscle caused by ketone bodies. Polaiologos & Felig (1976) have suggested that ketone bodies decrease alanine release from muscle: (a) by inhibition of glycolysis that decrease the pyruvate available for transamination with amino acids or (b) by inhibition of branched-chain amino acids catabolism. A number of workers have pointed out that the carbon skeleton of analine is formed also from some amino acids by the following route: after transamination, the keto acid skeleton is oxidized to Krebs cycle intermediates and malate formed in the cycle may be converted to pyruvate in reaction catalyzed by malic enzyme (Davis & Bremer. 1973: Spydevold ef al.. 1976; Goldstein & Newsholme. 1976; Garber er ul,, 1976: Lee & Davis, 1979). It seems likely that the effect of ketone bodies on alanine release from skeletal muscle could be explained by a direct inhibition of NADP-linked mitochondrial malic enzyme by acetyl-CoA (formed from ketone bodies). The data published recently (Swierczyriski et ctl.. 1980) as well as the results presented in this paper justify the assumption that during progressive starvation the formation of muscle alanine for gluconeogenesis may be partially dependent on the supply of pyruvate generated from malate produced from the oxidation of some amino acids. In this sequence of reactions the conversion of malate to pyruvate catalyzed by mitochondrial malic enzyme may be one of the most important regulatory steps. Stimulation of this malic enzyme by succinate or fumarate and inhibition by acetyl-CoA could be physiological regulatory factors. Although the metabolic role of maiic enzyme in skeletal muscle remains obscure. the present results provide further evidence that malic enzyme is present both ir, the cytosol and in the mitochondria and that the two forms of the enzyme have different properties. To obtain precise information about the metabolic role of the two isozymes. further investigations are required. A~kno\~letlyuments---I am indebted to Professor M. iydowo for a critical reading and dlscubsion of the manuscript. I am grateful to Dr. J. Spqchaia for performing the get electrophoresis. This work %a5 supported by a grant from Polish Academy of Sciences within the project MR II 1.2.4. REFEREiWES ASAMI S.. INWE K.. MATSLMWO K.. MC,RA~.HI A. & AUZAWA T. (19?9) NADP-malic enzvme from maize leaf: purification and properties. Ar,,f~s ~j~~~.~,~~~~. ~i~~~~i~.~.194, 503 510. BARTHOLOMEK.. BRDI(.ZL;A D. G. & PFTT~ D. (1972) Purification and properties of extra- and mirochondrial malate dehydrogenase INADP; decarboxyiating~ from pig heart. Hop~+Sq+~ Z. ph~i<$. Chrm. 353, 14X71495. BERNSTINF E. G. (1979) Genetic control of mitochondrial malic enzyme in mouse brain. J. hirll. Cl~tn. 254, X3 X7. BRANWIN P. C. & VAF: BOEKEL-MOL. T. N. (1973) Properties of purified malic enzyme in relation to crassulacean acid metabolism. Eur. J. Bio&m. 35. 62.--69.
BRDICZKAD. & PET‘TED. (1971) In&a- and extramitochondrial isozvmes of NADP malate dehydrogenase. Eur. J. Biochem. i9. 546--55 1. DAVIS E. J. & BREMERJ. (1973) Studies with isolated surviving rat hearts. Interdependence of free amino acids and citric-acid-cycle intermediates. Eur. J. ~j#ch~~~. 38, 86-97.
FRENUL R. (1971) Bovine heart malic enzyme. Isolation and partial purification of a cytoplasmic and mitochondrial enzyme. 1. hiol. Chem. 246, 3069-3074. FRENKELR. (1972) Isolation and some properties of a cytosol and a mitochondrial malic enzyme from bovine brain, Archs Biochem. Biophps. 152, 136143. FRENKEL R. & COBO-FRENKEI.A. (1973) Differential characteristics of the cytosol and mitochondrial isozymes of malic enzyme from bovine brain. Effects of dicarboxylic acids and sulfhydryf reagents. Archs Biothem. Biophys.
GARBERA. J., KARL 1. E. & KIPNIS D. M. (1976) Alanaine and glutamme synthesis and release from skeletal muscle. The precursor role of amino acids in alanine and glutamine synthesis. J. hiol. Chem. 251, 836843. GOLDSTEINL. & NEWSHOLME E. A. (1976) The formation of alanine from amino acids in diaphragm muscle of the rat. Biochem. J. 154, 555-558. HENDERSON N. S. (1966) Isozymes and genetic control of NADP-malate dehydrogenase in mice. A&s B&hem. Biophys. 117, 28-33. ISOHASHIF.. SH~BAYAMA K.. MARUYAMAE.. AOKI Y. & WADA F. (1971) Immunochemical studies on malate dehydrogenase (decarboxylating) (NADP). B&him. biophys.
Actu 250, 14-24.
LEE S-H. & DAVIS E. J. (1979) Carboxylation and decarboxylation reactions. Anaplerotic flux and removal of citrate cycle intermediates in skeletal muscle. J. biol. Chem. 254,420-430. NCWSHOLME E. A. & WILLIAMST. (1978) The role of phos-
phoenolpyruvate carboxykinase in amino acid metabolism in muscle. Biockem. J. 176, 623-626. NOLTE J.. BRDICZKAD. & PETTF D. 11972) Intracellular distribution of phosphoenolpyruvate carboxylase and NADP malate dehydrogenase in different muscle types. Bi(}~hirn. bjt~p~z~.s.Acrrc. 284. 497.-507.
P~LAIOLO~OS G. & FELICE P. (1976) Effects of ketone bodies on amino acid metabolism in isolated rat diaphragm. Biochml. J. 154, 709-716. SIMPSONE. R. & ESTABR~OK R.
W. (1969) Mitochondrial malic enzyme. The source of reduced nicotjnamide adenine dinucleotide phosphate for steroid hydroxylation in bovine adrenal cortex mitochondrin. Arch5 Biodwm. Bmphyx. 129, 384395. SPECTORT. (1978) Refinement of the Coomassie Blue method of protein quantitation, A simple and linear spectrophotometric assay for 0.5-50 pg of protein. rlnctIyt. Biochem.
SPYDEVOLL? 0.. DAVISE. J. & BREMI:RJ. (1976) Replenishment and depletion of citric acid cycle intermediates in skeletal muscle. Indication of pyruvate carboxylation. Eur. J. B&hem. 71, 155-165. SWIERCZY~~SKI J.. ALEKSANDROWICZ 2. & ~YDOWO M. (1975) Effect of some steroids and r-tocopherol on cytochrome c induced extramitochondrial NADH oxidation by human and rat skeletal muscle mitochondr~a. Inr J. Biachem.
6, 757 763.
SWIERCZY~~SKI J.. STANKIEW~LA.. &SI.OWSKI P. & ALEKSANDROWICZ A. (1980) Isolation and regulatory properties of mitochondrial malic enzyme from rat skeletal muscle. Biochim. b~~ph~.~. Acra 612, 1llO. SWIERCZY~~SKI J. (1980) Purification and some properties of extramitochondrial malic enzyme from rat skeletal muscle. Eiochim. hiophys. Acra 616, 10-21. SWIERCZY~~SKI J. (1981) Role of malic enzyme in pyruvate synthesis in the skeletal muscle. Ann. Acad. Med. GeBan. In press.