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     

The Oxidation of Malate by Isolated Turnip (Brassica napus L.) Mitochondria: II. THE MALATE-OXIDIZING ENZYMES, NUMBER AND LOCATION

The enzymes of malate oxidation in turnip mitochondria havebeen partially purified and some of their properties studied.Turnips contain a cytoplasmic malate dehydrogenase and two mito-chondrialmalate dehydrogenases. These are all distinct isoenzymes withdifferent immunblogical properties but similar molecular weights.The K_m for malate is relatively high (8.3 mM) in the mito-chondrialenzymes. One of the mitochondrial enzymes is located in thematrix while the other is membrane-bound but within the matrixcompartment. This latter enzyme, which retains its NAD and activitywhen submitochondrial particles are prepared, is responsiblefor the first phase of malate oxidation in submitochondrialparticles. Two malic enzymes were isolated: one, an NADP enzyme, is a minorcomponent and was not studied further; the other, immunologicallydistinct from the malate dehydrogenases, is probably locatedin the matrix compartment. The K_m for malate oxidation (1.4mM) is relatively low. This malic enzyme which apparently lacksOAA decarboxylase activity is NAD-specific but is unstable.The possibility of multiple malic enzymes is discussed. Themalic enzymes are responsible for the second NAD-requiring phaseof malate oxidation in submitochondrial particles.     

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.     

The mitochondrial malic enzymes. I. Submitochondrial localization and purification and properties of the NAD(P)+-dependent enzyme from adrenal cortex.

Rat and calf adrenal cortex homogenates were found to contain three different malic enzymes. Two were strictly NADP+-dependent and were localized, one each, in the cytosol and the mitochondrial fractions, respectively. These two enzymes appear to be identical to those described by Simpson and Estabrook (Simpson, E. R., and Estabrook, R. W. (1969) Arch. Biochem. Biophys. 129, 384-395). The third was NAD(P)+-linked and was present in the mitochondrial fraction only. All three malic enzymes separated as distinct bands during electrophoresis on 5 percent polyacrylamide slab gels at pH 9.0. Marker enzymes and the mitochondrial malic enzymes migrated together in intact mitochondria during sucrose density gradient centrifugations despite changes in the equilibrium position of the mitochondria promoted by energy-dependent calcium phosphate accumulation. In adrenal cortex mitochondria subfractionated by the method of Sottocasa et al. (SOTTOCASA, G.L., KUYLENSTIERNA, B., ERNSTER, L., and BERGSTAND, A. (1967) J. Cell Biol. 32, 415-438), both malic enzymes were associated with the inner membrane-matrix space. Sonication solubilized the two malic enzymes along with the matrix space marker enzymes. The NAD(P)+-dependent malic enzyme was purified 100-fold from calf adrenal cortex mitochondria. The final preparation was free of malic dehydrogenase, fumarase, the strictly NADP+-linked malic enzyme and adenylate kinase. Either Mn24 orMg2+ was required for activity and 1 mol of pyruvate was formed for each mole of NAD+ and NADP+ reduced. The pH optima with NAD+ and NADP+ were 6.5 tp 7.0 and 6.0 to 6.5, respectively. Michaelis-Menten kinetics were observed on the alkaline side. Fumarate, succinate, and isocitrate were positive and ATP and ADP were negative modulators of the regulatory enzyme. The modulators did not influence the stoichiometry and they were not metabolized during the reaction. Under Vmax conditions the ratios for the rate of NAD+:NADP+ reduction were 1.76 and 1.15 at pH 7.4 and 6.0, respectively. The apparent Michaelis constants also differed depending on the pH and the coenzyme. At pH 7.4 (in the presence of 5 mM fumarate) and at pH 6.0 (no fumarate) the Km values for (-)-malate, NAD+, and Mn2+ were 1.7, 0.16, and 0.15 mM, and 0.31, 0.06, and 0.09 mM, respectively. At pH 7.4 (5MM fumarate) and pH 6.0 (no fumarate), the Km values for (-)-malate, NADP+, and Mn2+ were 6.5, 0.62, and 0.59 mM, and 0.68. 0.12, and 0.31 mM, respectively. The apparent Ki values for ATP with NAD+ and NADP+ as coenzyme were 0.42 and 0.27 mM, respectively.     

Induction of hepatic mitochondrial glycerophosphate dehydrogenase in rats by dehydroepiandrosterone.

Feeding the thermogenic steroid, 5-androsten-3 beta-ol-17-one (dehydroepiandrosterone, DHEA) in the diet of rats induced the synthesis of liver mitochondrial sn-glycerol 3-phosphate dehydrogenase to levels three to five times that of control rats within 7 days. The previously reported enhancement of liver cytosolic malic enzyme was confirmed. The induction of both enzymes was detectable at 0.01% DHEA in the diet, reached plateau stimulation at 0.1 to 0.2%, and was completely blocked by simultaneous treatment with actinomycin D. Feeding DHEA caused smaller, but statistically significant increases of liver cytosolic lactate, sn-glycerol 3-phosphate, and isocitrate (NADP(+)-linked) dehydrogenases but not of malate or glucose 6-phosphate dehydrogenases. The capability of DHEA to enhance mitochondrial glycerophosphate dehydrogenase and malic enzyme was influenced by the thyroid status of the rats; was smallest in thyroidectomized rats and highest in rats treated with triiodothyronine. 5-Androsten-3 beta,17 beta-diol and 5-androsten-3 beta-ol-7,17-dione were as effective as DHEA in enhancing the liver mitochondrial glycerophosphate dehydrogenase and malic enzyme. Administering compounds that induce the formation of cytochrome P450 enzymes enhanced liver malic enzyme activity but not that of mitochondrial glycerophosphate dehydrogenase. Arochlor 1254 and 3-methylcholanthrene also increased the response of malic enzyme to DHEA feeding.     

Genetic variation of the cytoplasmic and mitochondrial malic enzymes in the monkey Macaca nemestrina

Electrophoretic variation of both the cytoplasmic and mitochondrial forms of the malic enzyme is described in Macaca nemestrina. Pedigree analysis of the observed phenotypes demonstrates that the two subcellular forms of the malic enzyme are genetically independent. The identity of the electrophoretic phenotypes in brain, heart muscle, liver, kidney, adrenal, and spleen from any given individual shows that each subcellular form is determined by the same genetic locus in a wide variety of tissues. After separation by ion exchange chromatography, the cytoplasmic and mitochondrial malic enzymes were shown to be distinct in their heat stability and K _m for malate, but no significant differences were found among the variants of the cytoplasmic enzyme or among the variants of the mitochondrial enzyme. It is possible that the polymorphism of the mitochondrial malic enzyme is selectively neutral.This study was supported by grant GM-15253 from the National Institutes of Health. One of us (G.S.O.) was a Special Fellow, U.S. Public Health Service (5F3 HD 43, 122-02); Fellow, National Genetics Foundation.     

Malic enzymes of salmon trout heart mitochondria: separation and some physicochemical properties of NAD-preferring and NADP-specific enzymes

Mitochondria isolated from the heart of the Baltic salmon trout Salmo trutta contain two distinct malic enzymes. One of these enzymes (NAD-preferring malic enzyme) catalyses the oxidative decarboxylation of malate in the presence of either NAD or NADP. The specific activity with NAD was six times that with NADP as coenzyme. The second enzyme is specific for NADP. These two malic enzymes have been separated by: ion exchange chromatography of DEAE-Sephacel, affinity chromatography on 2',5'ADP-Sepharose 4B, gel filtration on Sephacryl S-300 and polyacrylamide gel electrophoresis. The mol. wts of the two native malic enzymes determined by gel filtration were found to be 280,000 and 190,000 for NAD-preferring and NADP-specific malic enzyme, respectively. Chromatofocusing revealed the isoelectric points of the two enzymes at pH 5.45 and 5.85 for NAD-preferring and NADP-specific malic enzyme, respectively.     

Mitochondrial malate dehydrogenase and malic enzyme: Mendelian inherited electrophoretic variants in the mouse

Malate dehydrogenase and malic enzyme each possess supernatant and mitochondrial molecular forms which are structurally and genetically independent. We describe electrophoretic variants of the mitochondrial enzymes of malate dehydrogenase and malic enzyme in mice. Progeny testing from genetic crosses indicated that the genes which code for mitochondrial malate dehydrogenase and malic enzyme were not inherited maternally but as independent unlinked nuclear autosomal genes. The locus for mitochondrial malic enzyme was located on linkage group I. Linkage analysis with a third mitochondrial enzyme marker, glutamic oxaloacetic transaminase, showed that the nuclear genes which code for the three mitochondrial enzymes were not closely linked to each other. This evidence suggests that clusters of nuclear genes coding for mitochondrial function are unlikely in mice.Supported by U.S. Public Health Service grants 5F2 HD-35,531 and GM-09966.     

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.     

Mitochondrial malic enzyme (ME2) in pancreatic islets of the human, rat and mouse and clonal insulinoma cells: Simple enzyme assay for mitochondrial malic enzyme 2

Despite interest in malic enzyme(ME)s in insulin cells, mitochondrial malic enzyme (ME2) has only been studied with estimates of mRNA or with mRNA knockdown. Because an mRNA’s level does not necessarily reflect the level of its cognate enzyme, we designed a simple spectrophotometric enzyme assay to measure ME2 activity of insulin cells by utilizing the distinct kinetic properties of ME2. Mitochondrial ME2 uses either NAD or NADP as a cofactor, has a high K_m for malate and is allosterically activated by fumarate and inhibited by ATP. Cytosolic ME (ME1) and the other mitochondrial ME (ME3) use only NADP as a cofactor and have lower K_ms for malate. The assay easily showed for the first time that substantial ME2 activity is present in pancreatic islets of humans, rats and mice and INS-1 832/13 cells. ME2’s presence was confirmed with immunoblotting. There was no evidence that ME3 is present in these tissues.     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Crystal structure of human mitochondrial NAD(P)+-dependent malic enzyme: a new class of oxidative decarboxylases.

BACKGROUND: Malic enzymes catalyze the oxidative decarboxylation of malate to pyruvate and CO2 with the concomitant reduction of NAD(P)+ to NAD(P)H. They are widely distributed in nature and have important biological functions. Human mitochondrial NAD(P)+-dependent malic enzyme (mNAD-ME) may have a crucial role in the metabolism of glutamine for energy production in rapidly dividing cells and tumors. Moreover, this isoform is unique among malic enzymes in that it is a cooperative enzyme, and its activity is controlled allosterically. RESULTS: The crystal structure of human mNAD-ME has been determined at 2.5 A resolution by the selenomethionyl multiwavelength anomalous diffraction method and refined to 2.1 A resolution. The structure of the monomer can be divided into four domains; the active site of the enzyme is located in a deep cleft at the interface between three of the domains. Three acidic residues (Glu255, Asp256 and Asp279) were identified as ligands for the divalent cation that is required for catalysis by malic enzymes. CONCLUSIONS: The structure reveals that malic enzymes belong to a new class of oxidative decarboxylases. The tetramer of the enzyme appears to be a dimer of dimers. The active site of each monomer is located far from the tetramer interface. The structure also shows the binding of a second NAD+ molecule in a pocket 35 A away from the active site. The natural ligand for this second binding site may be ATP, an allosteric inhibitor of the enzyme.     

The oxidation of tricarboxylic acid cycle intermediates, with particular reference to isocitrate, by intact mitochondria isolated from the liver of the American eel, Anguilla rostrata leSueur.

The oxidation of exogenously added substrates has been studied in intact liver mitochondria isolated from the American eel, Anguilla rostrata. These data, coupled to determinations of the activity and localization of critical tricarboxylic acid (TCA) cycle enzymes, have been used to propose a pathway for the eel liver TCA cycle. (1) Isocitric, α-ketoglutaric, succinic, and malic acids are oxidized at essentially equivalent rates by eel mitochondria, with normal ADP:O and respiratory control ratios. No oxidation of citric, oxaloacetic, or pyruvic acids was detected when added alone or with malate, although oxaloacetic acid + pyruvic acid was oxidized but at a much reduced rate. (2) Radioactively labeled isocitrate was incorporated into at least α-ketoglutaric, succinic, and malic acids, indicating the eel liver TCA cycle is normal between isocitrate and malate. (3) No activity of the NAD-linked isocitrate dehydrogenase (IDH) could be detected, but NADP-IDH activities were higher in the mitochondria than cytosolic fractions. An active NADPH:NAD transhydrogenase was localized to the mitochondrial compartment. (4) These data suggest an important role for the NADP-IDH:transhydrogenase enzyme couple in eel liver TCA cycle function, and a pathway incorporating these ideas is proposed.     

Guanylate cyclase: assay and properties of the particulate and supernatant enzymes in mouse parotid.

A new, very sensitive, rapid and reliable assay for guanylate cyclase has been established based on conversion of [32P]GTP to [32P]guanosine 3':5'-monophosphate and its separation on Dowex 50 and aluminium oxide columns. The optimum conditions for the assay of mouse parotid guanylate cyclase have been established and using this procedure the properties of the enzyme have been investigated. The enzyme was found in both the particulate and supernatant fractions. The particulate enzyme was activated 12-fold by Triton X-100 and the supernatant enzyme activity increased 2-fold. In the presence of detergent guanylate cyclase activity was distributed 85% in the particulate and 15% in the supernatant fractions, respectively. The particulate activity was localised in a plasma membrane fraction. Guanylate cyclase activity was also assayed in a wide variety of other tissues. In all cases enzymatic activity was found in both the particulate and supernatant fractions. The distribution varied with the tissue but only the intestinal mucosa had a greater proportion of total guanylate cyclase activity in the particulate fraction than the parotid. The two enzymes showed some similar properties. Their pH optima were pH 7.4, both enzymes were inhibited by ATP, dATP, dGTP and ITP, required Mn2+ for activity and plots of activity versus Mn2+ concentration were sigmoidal. However, in many properties the enzymes were dissimilar. The ratios of Mn2+ to GTP for optimum activity were 4 and 1.5 for the supernatant and plasma-bound enzymes, respectively. The slope of Hill plots for the supernatant enzyme with varying Mn2+ was 2. The particulate enzyme plots also had a slope of 2 at low Mn2+ concentration but at higher concentrations (above 0.7 mM) the Hill coefficient shifted abruptly to 4. Calcium ions reduced sigmoidicity of the kinetics lowering the Hill coefficient, activated the enzyme at all Mn2+ concentrations but had no effect on the Mn2+:GTP ratio with the supernatant enzyme while with the plasma membrane enzyme Ca2+ had no effect on the sigmoid form of the kinetics at low Mn2+ but prevented the shift to a greater Hill coefficient at higher Mn2+, inhibited the activity at low Mn2+ and shifted the Mn2+:GTP optimum ratio to 4. For the particulate enzyme plots of activity versus GTP concentration were sigmoid (n = 1.3), while the supernatant enzyme exhibited hyperbolic kinetics.     

Kinetic studies with cytosol and mitochondrial phosphoenolpyruvate carboxykinases

1. Measurements of Michaelis constants for oxaloacetate in the reaction catalysed by liver phosphoenolpyruvate carboxykinase give values much lower than previously reported. With Mg(2+) as bivalent cation, the Michaelis constant was approx. 2.5x10(-5)m whether the enzyme used was the mitochondrial phosphoenolpyruvate carboxykinase purified from sheep liver or chicken liver or the cytosol enzyme purified from rat liver or sheep liver. 2. When Mn(2+) replaced Mg(2+) in the reaction a lower Michaelis constant of 9x10(-6)m was found, but only with the mitochondrial enzymes. 3. With all enzymes malate at high concentration was a competitive inhibitor with respect to oxaloacetate when Mn(2+) was the added cation. With Mg(2+) the inhibition by malate was competitive with the mitochondrial enzymes and non-competitive with the cytosol enzymes.     

Intermediary carbohydrate metabolism in the adult filarial worm Setaria digitata.

Several key enzymes related to carbohydrate metabolism were assayed in Setaria digitata. In the cytosolic fraction pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malic enzyme, aspartate transaminase and alanine transaminase were found. Among the TCA cycle enzymes succinate dehydrogenase, fumarate reductase, fumarase (malate dehydration), malate dehydrogenase (malate oxidation and oxaloacetate reduction) and malic enzyme (malate decarboxylation) were detected in the mitochondrial fraction. Only reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase, NADH oxidase and NADH-cytochrome c reductase were found in the mitochondrial fraction. The significance of these results with respect to the metabolic capabilities of the worm are discussed.     

Effect of thyroidectomy on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis

The specific activities of testicular enzymes of the pyruvate/malate cycle involved in lipogenesis after thyroidectomy and thyroxine replacement were studied in prepubertal, pubertal and adult rats. Thyroidectomy induced testicular ATP citrate-lyase, malate dehydrogenase and malic enzyme activities and inhibited isocitrate dehydrogenase (NADP⁺) activity. Thyroxine treatment on thyroidectomized animals reverted all enzyme activities to normal. The result suggests that thyroid hormones have a differential effects on testicular enzymes of the pyruvate/malate cycle involved in lipogenesis.     

Malate Metabolism in Leaf Mitochondria from the Crassulacean Acid Metabolism Plant Kalanchoë blossfeldiana Poelln

The mechanisms and the controlling factors of malate oxidation by mitochondria from leaves of Kalanchoë blossfeldiana Poelln. plants performing Crassulacean acid metabolism were investigated using Percollpurified mitochondria. The effects of pH and of various cofactors (ATP, NAD⁺, coenzyme A) on malate dehydrogenase (EC 1.1.1.37) and malic enzyme (EC 1.1.1.39) solubilized from these mitochondria were examined. The crucial role of cofactor concentrations in the mitochondrial matrix on the pathways of malate oxidation is shown. The distribution of the electrons originating from malate between the different electron transport pathways and its consequence on the phosphorylation yield was studied. It was found that, depending on the electron transport pathway used, malate oxidation could yield from 3 to 0 ATP. Assayed under conditions of high reducing power and high energy charge, the ability of malic enzyme to feed electrons to the cyanide-resistant nonphosphorylating alternative pathway was found to be higher than that of other dehydrogenases linked to the functioning of the Krebs cycle (pyruvate dehydrogenase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase, succinate dehydrogenase). The physiological significance of such a functional relationship between malic enzyme activity and the nonphosphorylating alternative pathway is discussed in relation to Crassulacean acid metabolism.     

Malic enzyme in human liver. Intracellular distribution, purification and properties of cytosolic isozyme

In human liver, almost 90% of malic enzyme activity is located within the extramitochondrial compartment, and only approximately 10% in the mitochondrial fraction. Extramitochondrial malic enzyme has been isolated from the post-mitochondrial supernatant of human liver by (NH4)2SO4 fractionation, chromatography on DEAE-cellulose, ADP-Sepharose-4B and Sephacryl S-300 to apparent homogeneity, as judged from polyacrylamide gel electrophoresis. The specific activity of the purified enzyme was 56 mumol.min-1.mg protein-1, which corresponds to about 10,000-fold purification. The molecular mass of the native enzyme determined by gel filtration is 251 kDa. SDS/polyacrylamide gel electrophoresis showed one polypeptide band of molecular mass 63 kDa. Thus, it appears that the native protein is a tetramer composed of identical-molecular-mass subunits. The isoelectric point of the isolated enzyme was 5.65. The enzyme was shown to carboxylate pyruvate with at least the same rate as the forward reaction. The optimum pH for the carboxylation reaction was at pH 7.25 and that for the NADP-linked decarboxylation reaction varied with malate concentration. The Km values determined at pH 7.2 for malate and NADP were 120 microM and 9.2 microM, respectively. The Km values for pyruvate, NADPH and bicarbonate were 5.9 mM, 5.3 microM and 27.9 mM, respectively. The enzyme converted malate to pyruvate (at optimum pH 6.4) in the presence of 10 mM NAD at approximately 40% of the maximum rate with NADP. The Km values for malate and NAD were 0.96 mM and 4.6 mM, respectively. NAD-dependent decarboxylation reaction was not reversible. The purified human liver malic enzyme catalyzed decarboxylation of oxaloacetate and NADPH-linked reduction of pyruvate at about 1.3% and 5.4% of the maximum rate of NADP-linked oxidative decarboxylation of malate, respectively. The results indicate that malic enzyme from human liver exhibits similar properties to the enzyme from animal liver.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.