Malate utilization by a group D Streptococcus: physiological properties and purification of an inducible malic enzyme

Growth of Streptococcus faecalis in the presence of l-malate resulted in the induction of a "malic enzyme" [l-malate:nicotinamide adenine dinucleotide (NAD) oxidoreductase (decarboxylating), E.C. 1.1.1.39]. Synthesis of the malic enzyme did not appear to be subject to catabolite repression by intermediate products of glucose or fructose dissimilation. However, malate utilization was inhibited during growth in the presence of glucose or fructose. The purified enzyme was specific for malate as substrate and NAD as cofactor. Mn(+2) or Mg(+2) was required for optimal activity and NH(4)Cl stimulated the reaction rate. Several lines of indirect evidence suggested that the streptococcal malic enzyme was involved primarily with energy production and not biosynthesis.     

The activation of adrenal cortex mitochondrial malic enzyme by Ca2+ and Mg2+.

Adrenal cortex mitochondria prepared by a standard method do not exhibit malic enzyme activity. Addition of physiological concentrations of Ca2+ and Mg2+ enables these mitochondria to reduce added NADP+ by malate to form free NADPH. Half-maximum activation of the mitochondrial malic enzyme requires 0.3 mM Ca2+ and 1 mM Mg2+. Solubilized mitochondrial malic enzymes is independent of Ca2+ and has a K M of 0.2 mM for Mg2+. The Ca2+ effect is dependent on an initial period of active Ca2+ uptake which also causes other changes in respiratory properties similar to those observed with mitochondria from other tissues. After Ca2+ accumulation has taken place, free Ca2+, but not additional accumulation, is still required for malic enzyme activity. The requirement for Mg2+ can be met by Mn2+ (1 mM). This concentration of Mn2+ alone yielded only a slight activation of mitochondrial malic enzyme while higher concentrations of Mn2+ alone gave good activation of the mitochondrial malic enzy.e The NADPH generated by the Ca2+-Mg2+ activated malic enzyme effectively supports the 11beta-hydroxylation of deoxycorticosterone, whereas in the presence of malate, or malate plus Mg2+ but absence of Ca2+, the energy linked transhydrogenase supplies all the required NADPH. The activated malic enzyme appears to be more efficient than transhydrogenase in generating NADPH to support 11beta-hydroxylation. Cyanide and azide have been found to inhibit solubilized mitochondrial malic enzyme.     

Regulation of mitochondrial and cytosolic malic enzymes from cultured rat brain astrocytes

Malate has a number of key roles in the brain, including its function as a tricarboxylic acid (TCA) cycle intermediate, and as a participant in the malate-aspartate shuttle. In addition, malate is converted to pyruvate and CO₂ via malic enzyme and may participate in metabolic trafficking between astrocytes and neurons. We have previously demonstrated that malate is metabolized in at least two compartments of TCA cycle activity in astrocytes. Since malic enzyme contributes to the overall regulation of malate metabolism, we determined the activity and kinetics of the mitochondrial and cytosolic forms of this enzyme from cultured astrocytes. Malic enzyme activity measured at 37°C in the presence of 0.5 mM malate was 4.15±0.47 and 11.61±0.98 nmol/min/mg protein, in mitochondria and cytosol, respectively (mean±SEM, n=18–19). Malic enzyme activity was also measured in the presence of several endogenous compounds, which have been shown to alter intracellular malate metabolism in astrocytes, to determine if these compounds affected malic enzyme activity. Lactate inhibited cytosolic malic enzyme by a noncompetitive mechanism, but had no effect on the mitochondrial enzyme. -Ketoglutarate inhibited both cytosolic and mitochondrial malic enzymes by a partial noncompetitive mechanism. Citrate inhibited cytosolic malic enzyme competitively and inhibited mitochondrial malic enzyme noncompetitively at low concentrations of malate, but competitively at high concentrations of malate. Both glutamate and aspartate decreased the activity of mitochondrial malic enzyme, but also increased the affinity of the enzyme for malate. The results demonstrate that mitochondrial and cytosolic malic enzymes have different kinetic parameters and are regulated differently by endogenous compounds previously shown to alter malate metabolism in astrocytes. We propose that malic enzyme in brain has an important role in the complete oxidation of anaplerotic compounds for energy.These data were presented in part at the meeting of the American Society for Neurochemistry in Richmond, Virginia, March 1993     

Crithidia fasciculata: regulation of aerobic fermentation by malic enzyme

Aerobic fermentation, described in many protozoan organisms, was investigated using the nonpathogenic protozoan Crithidia fasciculata. The major end-products of this process were organic acids, particularly succinic acid. Inhibition of malic enzyme (EC 1.1.1.40) (l-malate: NADP oxidoreductase (decarboxylating) appeared to be integral to the aerobic fermentative process; the enzyme was inhibited in a cumulative manner by oxalacetate, acetyl-coenzyme A and oxalate. Inhibition by oxalate was noncompetitive with respect to both substrate and coenzyme. The inhibition of malic enzyme by oxalacetate and acetyl-coenzyme A appeared to provide a mechanism for the diversion of carbon from oxalacetate to succinate via the fumarase, thereby avoiding recycling to pyruvate. This was corroborated by the demonstration of an inverse relationship between the concentrations of pyruvate and succinate elaborated by this organism.     

Nicotinamide-adenine dinucleotide-linked "malic" enzyme in flight muscle of the tse-tse fly (Glossina) and other insects.

1. A high activity of NAD-linked "malic" enzyme was found in homogenates of flight muscle of different species of tse-tse fly (Glossina). The activity was the same as, or higher than, that of malate dehydrogenase and more than 20-fold that of NADP-linked "malic" enzyme. A similar enzyme was found in the flight muscle of all other insects investigated, but at much lower activities. 2. ACa2+-stimulated oxaloacetate decarboxylase activity was present in all insect flight-muscle preparations investigated, in constant proportion to the NAD-linked "malic" enzyme. 3. A partial purification of the NAD-linked "malic" enzyme from Glossina was effected by DEAE-cellulose chromatography, which separated the enzyme from malate dehydrogenase and NADP-linked "malic" enzyme, but not from oxaloacetate decarboxylase. 4. The intracellular localization of the NAD-linked "malic" enzyme was predominantly mitochondrial; latency studies suggested a localization in the mitochondrial matrix space. 5. Studies on the partially purified enzyme demonstrated that it had a pH optimum between 7.6 and 7.9. It required Mg2+ or Mn2+ for activity; Ca2+ was not effective. The maximum rate was the same with either cation, but the concentration of Mn2+ required was 100 times less than that of Mg2+. Acitivity with NADP was only 1-3% of that with NAD, unless very high (greater than 10mM) concentrations of Mn2+ were present. 6. It is suggested that the NAD-linked "malic" enzyme functions in the proline-oxidation pathway predominant in tse-tse fly flight muscle.     

Changes of malic enzyme activity in the developing rat brain are due to both the increase of mitochondrial protein content and the increase of specific activity.

1. The pattern of NADP-linked malic enzyme activity estimated in the whole brain homogenate did not parallel that found in liver of developing rat. 2. Studies on intracellular distribution of malic enzyme in brain showed that the mitochondrial enzyme increased about three-fold between 10th and 40th day of life. Thereafter, a slow gradual increase to the adult level was observed. 3. The extramitochondrial malic enzyme from brain, like the liver enzyme, increased at the time of weaning, although to a lesser extent. At day 5 the brain malic enzyme was equally distributed between mitochondria and cytosol. 4. During the postnatal development, the contribution of the mitochondrial malic enzyme in the total activity was increasing, reaching the value approx. 80% at day 150 after birth. 5. The increase with age of the malic enzyme specific activity was observed in both synaptosomal and non-synaptosomal mitochondria, the changes in the last fraction being more pronounced. 6. The activity of citrate synthase developed markedly between 10-40 postnatal days, increasing about five-fold, while the specific activity of the enzyme did change neither in the synaptosomal nor in non-synaptosomal mitochondria at this period. 7. We conclude that the changes in malic enzyme activity in the developing rat brain are mainly due both to the increase of mitochondrial protein content and to the increase of specific activity of the mitochondrial malic enzyme.     

Role and location of NAD malic enzyme in thermogenic tissues of Araceae

This work was done to discover how those nonphotosynthetic tissues of the Araceae that become thermogenic release, as CO2, carbon recently fixed by phosphoenolpyruvate carboxylase. Extracts of clubs of the spadix of Arum maculatum showed no activity for phosphoenolpyruvate carboxykinase and low activities of NADP malic enzyme. NAD malic enzyme activity in the above extracts and in those of thermogenic tissues of other Araceae was appreciable. Analysis of homogenates of clubs of Typhonium giraldii by differential centrifugation and sucrose gradients showed that NAD malic enzyme was confined to mitochondria. Centrifugation of mitochondria after freezing and thawing left all the NAD malic enzyme in the supernatant. NAD malic enzyme in isolated, intact mitochondria was completely latent, and was completely protected from exogenous trypsin. The responses of this latency and protection to different concentrations of Triton X-100 suggested that none of the NAD malic enzyme was accessible from either the outside or the intermembrane space of the mitochondria. Treatment of excised clubs of A. maculatum with 2-N-butylmalonate largely prevented the development of the rapid respiration responsible for thermogenesis, and severely inhibited dark fixation of 14CO2. The conclusion is that in mature clubs of the Araceae phosphoenolpyruvate is converted to malate in the cytosol by phosphoenolpyruvate carboxylase and NAD malate dehydrogenase, and that this malate then enters the mitochondrial matrix where it is converted to pyruvate by NAD malic enzyme.     

Pigeon liver malic enzyme: Involvement of an arginyl residue at the binding site for malate and its analogs

Treatment of malic enzyme with arginine-specific reagents phenylglyoxal or 2,3-butanedione results in pseudo-first-order loss of oxidative decarboxylase activity. Inactivation by phenylglyoxal is completely prevented by saturating concentrations of NADP⁺, Mn²⁺, and substrate analog hydroxymalonate. Double log plots of pseudo-first-order rate constant versus concentration yield straight lines with identical slopes of unity for both reagents, suggesting that reaction of one molecule of reagent per active site is associated with activity loss. In parallel experiments, complete inactivation is accompanied by the incorporation of four [¹⁴C]phenylglyoxal molecules, and the loss of two arginyl residues per enzyme subunit, as determined by the colorimetric method of Yamasaki et al (R. B. Yamasaki, D. A. Shimer, and R. E. Feeney (1981) Anal. Biochem., 14, 220–226). These results confirm a 2:1 ratio for the reaction between phenylglyoxal and arginine (K. Takahashi (1968) J. Biol. Chem., 243, 6171–6179) and yield a stoichiometry of two arginine residues reacted per subunit for complete inactivation, of which one is essential for enzyme activity as determined by the statistical method of Tsou (C. L. Tsou (1962) Acta Biochim. Biophys. Sinica, 2, 203–211) and the Ray and Koshland analysis (W. J. Ray and D. E. Koshland (1961) J. Biol. Chem., 236, 1973–1979). Amino acid analysis of butanedione-modified enzyme also shows loss of arginyl residues, without significant decrease in other amino acids. Modification by phenylglyoxal does not significantly affect the affinity of this enzyme for NADPH. Binding of l-malate and its dicarboxylic acid analogs oxalate and tartronate is abolished upon modification, as is binding of the monocarboxylic acid α-hydroxybutyrate. The latter result indicates binding of the C-1 carboxyl group of the substrate to an arginyl residue on the enzyme.     

Partial Purification and Properties of Human Brain Aldehyde Dehydrogenases

Acetaldehyde and biogenic aldehydes were used as substrates to investigate the subcellular distribution of aldehyde dehydrogenase activity in autopsied human brain. With 10 microM acetaldehyde as substrate, over 50% of the total activity was found in the mitochondrial fraction and 38% was associated with the cytosol. However, with 4 microM 3,4-dihydroxyphenylacetaldehyde and 10 microM indoleacetaldehyde as substrates, 40-50% of the total activity was found in the soluble fraction, the mitochondrial fraction accounting for only 15-30% of the total activity. These data suggested the presence of distinct aldehyde dehydrogenase isozymes in the different compartments. The mitochondrial and cytosolic fractions were, therefore, subjected to salt fractionation and ion-exchange chromatography to purify further the isozymes present in both fractions. The kinetic data on the partially purified isozymes revealed the presence of a low Km isozyme in both the mitochondria and the cytosol, with Km values for acetaldehyde of 1.7 microM and 10.2 microM, respectively. However, the cytosolic isozyme exhibited lower Km values for the biogenic aldehydes. Both isozymes were activated by Mg2+ and Ca2+ in phosphate buffers (pH 7.4). Also, high Km isozymes were found in the mitochondria and in the microsomes.     

Mitochondrial malic enzyme activity is much higher in mitochondria from cortical synaptic terminals compared with mitochondria from primary cultures of cortical neurons or cerebellar granule cells

Most of the malic enzyme activity in the brain is found in the mitochondria. This isozyme may have a key role in the pyruvate recycling pathway which utilizes dicarboxylic acids and substrates such as glutamine to provide pyruvate to maintain TCA cycle activity when glucose and lactate are low. In the present study we determined the activity and kinetics of malic enzyme in two subfractions of mitochondria isolated from cortical synaptic terminals, as well as the activity and kinetics in mitochondria isolated from primary cultures of cortical neurons and cerebellar granule cells. The synaptic mitochondrial fractions had very high mitochondrial malic enzyme (mME) activity with a Km and a Vmax of 0.37 mM and 32.6 nmol/min/mg protein and 0.29 mM and 22.4 nmol/min mg protein, for the SM2 and SM1 fractions, respectively. The Km and Vmax for malic enzyme activity in mitochondria isolated from cortical neurons was 0.10 mM and 1.4 nmol/min/mg protein and from cerebellar granule cells was 0.16 mM and 5.2 nmol/min/mg protein. These data show that mME activity is highly enriched in cortical synaptic mitochondria compared to mitochondria from cultured cortical neurons. The activity of mME in cerebellar granule cells is of the same magnitude as astrocyte mitochondria. The extremely high activity of mME in synaptic mitochondria is consistent with a role for mME in the pyruvate recycling pathway, and a function in maintaining the intramitochondrial reduced glutathione in synaptic terminals.     

Malic enzyme activity and related Biochemical aspects during ripening of γ-irradiated mango fruit

The increase in malic enzyme (l-malate: NADP⁺ oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) activity, usually observed during the r     

Subcellular distribution of aminotransferases, and pyruvate branch point enzymes in gill tissue from four bivalves

Aspartate aminotransferase (AAT), alanine aminotransferase (ALAT), malic enzyme (ME), malate dehydrogenase (MDH), pyruvate kinase (PK), and phosphoenolpyruvate carboxykinase (PEPCK) activities in cytosolic and mitochondrial fractions of gill tissue from Modiolus demissus (ribbed mussel), Mytilus edulis (sea mussel), Crassostrea virginica (oyster) and Mercenaria mercenaria (quahog) were determined using enzyme assay and starch gel electrophoresis combined with subcellular fractionation. AAT showed distinct mitochondrial and cytosolic isozymes in gills of all these animals. Although ALAT showed distinct mitochondrial and cytosolic isozymes in the gills of oysters, sea mussels and quahogs, only the mitochondrial ALAT was evident in ribbed mussel gill tissue. PK and PEPCK were cytosolic in all these preparations. ME was found only in the mitochondrial fraction of ribbed mussel and quahog gill tissue whereas sea mussel gills showed distinct cytosolic and mitochondrial ME isozymes. With oyster gills, the "cytosolic ME" was electrophoretically identical to the mitochondrial ME indicating that in vivo, the ME is probably mitochondrial. MDH showed distinct cytosolic and mitochondrial isozymes in all bivalve gills tested.     

Clofibrate feeding increases cytoplasmic but not mitochondrial malic enzyme activity in rat kidney cortex

Administration of clofibrate for 21 days to rats increased the malic enzyme activity in the kidney cortex by about 80 per cent. This effect seems to be specific since the drug did not alter significantly the activity either of lactate dehydrogenase, citrate synthase or total mitochondrial protein content in this organ. The increase in activity of malic enzyme in the 13,000 g supernatant (extramitochondrial) fraction in rats treated with the drug was about 80 per cent, whereas in the pellet (mitochondrial fraction) it was about 40 per cent. The specific activity of malic enzyme in the kidney cortex cytosol from clofibrate-treated rats was about twice that in controls. In contrast clofibrate treatment did not affect its specific activity in isolated mitochondria. Calculations showed that 0.57 and 0.53 mumoles min-1 g-1 wet tissue of mitochondrial malic enzyme was obtained in control and clofibrate-treated rats respectively. Thus, clofibrate feeding increases the amount of cytoplasmic but not mitochondrial malic enzyme activity.     

The subcellular location of isozymes of NADP-isocitrate dehydrogenase in tissues from pig, ox and rat

Antibodies against purified NADP-isocitrate dehydrogenase from pig liver cytosol and pig heart were raised in rabbits. The purified enzymes from these sources are different proteins, as demonstrated by differences in electrophoretic mobility and absence of crossreactivity by immunotitration and immunodiffusion. The NADP-isocitrate dehydrogenase in the soluble supernatant homogenate fraction from pig liver, kidney cortex, brain and erythrocyte hemolyzate was identical with the purified enzyme from pig liver cytosol, as determined by electrophoretic mobility and immunological techniques. The enzyme in extracts of mitochondria from pig heart, kidney, liver and brain was identical with the purified pig heart enzyme by the same criteria. However, the 'mitochondrial' isozyme was the major component also in the soluble supernatant fraction of pig heart homogenate. The 'cytosolic' isozyme accounted for only 1-2% of total NADP-isocitrate dehydrogenase in pig heart, as determined by separation of the isozymes with agarose gel electrophoresis and immunotitration. The mitochondrial isozyme was also the predominant NADP-isocitrate dehydrogenase in porcine skeletal muscle. The ratio of cytosolic/mitochondrial isozyme for porcine whole tissue extract, determined by immunotitration, was about 2 for liver and 1 for kidney cortex and brain. The distribution of isozymes in cell homogenate fractions from ox and rat tissues corresponded to that observed in organs of porcine origin. The mitochondrial and cytosolic isozymes from ox and rat tissues exhibited crossreactivity with the antibodies against the pig heart and pig liver cytosol enzyme, respectively, and the electrophoretic migration patterns were similar qualitatively to those found for the isozymes in porcine tissues. Nevertheless, there were species specific differences in the characteristics of each of the corresponding isozymes. NAD-isocitrate dehydrogenase was not inhibited by the antibodies, confirming that the protein is distinct from that of either isozyme of NADP-isocitrate dehydrogenase.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

Fumarate and cytosolic pH as modulators of the synthesis or consumption of C4 organic acids through NADP-malic enzyme in Arabidopsis thaliana

Arabidopsis thaliana is a plant species that accumulates high levels of organic acids and uses them as carbon, energy and reducing power sources. Among the enzymes that metabolize these compounds, one of the most important ones is malic enzyme (ME). A. thaliana contains four malic enzymes (NADP-ME 1–4) to catalyze the reversible oxidative decarboxylation of malate in the presence of NADP. NADP-ME2 is the only one located in the cell cytosol of all Arabidopsis organs providing most of the total NADP-ME activity. In the present work, the regulation of this key enzyme by fumarate was investigated by kinetic assays, structural analysis and a site-directed mutagenesis approach. The final effect of this metabolite on NADP-ME2 forward activity not only depends on fumarate and substrate concentrations but also on the pH of the reaction medium. Fumarate produced an increase in NADP-ME2 activity by binding to an allosteric site. However at higher concentrations, fumarate caused a competitive inhibition, excluding the substrate malate from binding to the active site. The characterization of ME2-R115A mutant, which is not activated by fumarate, confirms this hypothesis. In addition, the reverse reaction (reductive carboxylation of pyruvate) is also modulated by fumarate, but in a different way. The results indicate pH-dependence of the fumarate modulation with opposite behavior on the two activities analyzed. Thereby, the coordinated action of fumarate over the direct and reverse reactions would allow a precise and specific modulation of the metabolic flux through this enzyme, leading to the synthesis or degradation of C₄ compounds under certain conditions. Thus, the physiological context might be exerting an accurate control of ME activity in planta, through changes in metabolite and substrate concentrations and cytosolic pH.     

Human placental aldehyde dehydrogenase. Subcellular distribution and properties

Freshly obtained human term placentae were subjected to subcellular fractionation to study the localization of NAD-dependent aldehyde dehydrogenases. Optimal conditions for the cross-contamination-free subcellular fractionation were standardized as judged by the presence or the absence of appropriate marker enzymes. Two distinct isozymes, aldehyde dehydrogenase I and II, were detected in placental extracts after isoelectric focusing on polyacrylamide gels. Based on a placental wet weight, about 80% of the total aldehyde dehydrogenase activity was found in the cytosolic acid and about 10% in the mitochondrial fraction. The soluble fraction (cytosol) contained predominantly aldehyde dehydrogenase II which has a relatively high Km (9 mmol/l) for acetaldehyde and is strongly inhibited by disulfiram. The results indicate that cytosol is the main site for acetaldehyde oxidation, but the enzyme activity is too slow to prevent the placental passage of normal concentrations of blood acetaldehyde (less than 1 mumol/l) produced by maternal ethanol metabolism.     

Purification and characterization of cytosol-specific phosphoenolpyruvate carboxykinase from chicken liver

Phosphoenolpyruvate carboxykinase of chicken liver cytosol was purified to homogeneity by procedures including affinity chromatography with GTP as a ligand. The purified enzyme showed a molecular weight of 68,000 on gel electrophoresis in the presence of dodecyl sulfate. Comparative studies on this enzyme and its isozyme purified from chicken liver mitochondria were performed. As regards amino acid composition, the cytosolic enzyme was quite different from the mitochondrial enzyme, but was rather similar to rat liver cytosolic phosphoenolpyruvate carboxykinase. Specific activities of the cytosolic enzyme were 30-100% higher than those of the mitochondrial enzyme for oxaloacetate-CO2 exchange, oxaloacetate decarboxylation, and phosphoenolpyruvate carboxylation reactions, though the relative rates of the activities were similar, decreasing in the order given. Apparent Michaelis constants for oxaloacetate in the oxaloacetate decarboxylation reaction were 11.6 and 17.9 microM for the cytosolic and the mitochondrial enzyme, respectively, but the values for GTP, GDP, phosphoenolpyruvate, and CO2 in the oxaloacetate decarboxylation and phosphoenolpyruvate carboxylation reactions were 1.3-2.2 times higher for the cytosolic enzyme than for the mitochondrial enzyme. Thus, the fundamental catalytic properties of the chicken liver phosphoenolpyruvate carboxykinase isozymes were rather similar, despite the marked difference in amino acid compositions.     

Kinetics and regulation of hepatoma mitochondrial NAD(P) malic enzyme.

Kinetic studies of Morris 7777 hepatoma mitochondrial NAD(P) malic enzyme were consistent with an ordered mechanism where NAD adds to the enzyme before malate and dissociation of NADH from the enzyme is rate-limiting. In addition to its active site, malate apparently also associates with a lower affinity with an activator site. The activator fumarate competes with malate at the activator site and facilitates dissociation of NADH from the enzyme. The ratio of NAD(P) malic enzyme to malate dehydrogenase activity in the hepatoma mitochondrial extract was found to be too low, even in the presence of known inhibitors of malate dehydrogenase, to account for the known ability of NAD(P) malic enzyme to intercept exogenous malate from malate dehydrogenase in intact tumor mitochondria (Moreadith, R.W., and Lehninger, A.L. (1984) J. Biol. Chem. 259, 6215-6221). However, NAD(P) malic enzyme may be able to intercept exogenous malate because according to the present results, it can associate with the pyruvate dehydrogenase complex, which could localize NAD(P) malic enzyme in the vicinity of the inner mitochondrial membrane. The activity levels of some key metabolic enzymes were found to be different in Morris 7777 mitochondria than in liver or mitochondria of other rapidly dividing tumors. These results are discussed in terms of differences among tumors in their ability to utilize malate, glutamate, and citrate as respiratory fuels.