Purification and properties of cytosolic malic enzyme from human skeletal muscle

1. An NADP+-dependent malic enzyme was purified 7940-fold from the cytosolic fraction of human skeletal muscle with a final yield of 55.8% and a specific activity of 38.91 units/mg of protein. 2. The purification to homogeneity was achieved by ammonium sulfate fractionation, DEAE-Sepharose chromatography, affinity chromatography on NADP+-Agarose, gel filtration on Sephacryl S-300 and rechromatography on the affinity column. 3. Either Mn2+ or Mg2+ was required for activity: the pH optima with Mn2+ and Mg2+ were 8.1 and 7.5, respectively. The enzyme showed Michaelis-Menten kinetics. At pH 7.5 the apparent Km values with Mn2+ and Mg2+ for L-malate and NADP+ were 0.246 mM and 5.8 microM, and 0.304 mM and 5.8 microM, respectively. The Km values with Mn2+ for pyruvate, NADPH and bicarbonate were 8.6 mM, 6.1 microM and 22.2 mM, respectively. 4. The enzyme was also able to decarboxylate malate in the presence of NAD+. At pH 7.5 the reaction rate was approximately 10% of the rate in the presence of NADP+, with a Km value for NAD+ of 13.9 mM. 5. The following physical parameters were established: s0(20.w) = 10.48, Stokes' radius = 5.61 nm, pI = 5.72 Mr of the dissociated enzyme = 61,800. The estimates of the native apparent Mr yielded a value of 313,000 upon gel filtration, and 255,400 with f/fo = 1.33 by combining the chromatographic data with the sedimentation measurements. 6. The electron microscopy analysis of the uranyl acetate-stained enzyme revealed a tetrameric structure. 7. Investigations to detect sugar moieties indicated that the enzyme contains carbohydrate side chains, a property not previously reported for any other malic enzyme.     

Malic enzyme from archaebacterium Sulfolobus solfataricus. Purification, structure, and kinetic properties

An NADP-preferring malic enzyme ((S)-malate:NADP oxidoreductase (oxalacetate-decarboxylating) EC 1.1.1.40) with a specific activity of 36.6 units per mg of protein at 60 degrees C and an isoelectric point of 5.1 was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus, strain MT-4. The purification procedure employed ion exchange chromatography, ammonium sulfate fractionation, affinity chromatography, and gel filtration. Molecular weight determinations demonstrated that the enzyme was a dimer of Mr 105,000 +/- 2,000 with apparently identical Mr 49,000 +/- 1,500 subunits. Amino acid composition of S. solfataricus enzyme was determined and found to be significantly higher in tryptophan content than the malic enzyme from Escherichia coli. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, S. solfataricus malic enzyme was able to catalyze the decarboxylation of oxalacetate. The enzyme absolutely required divalent metal cations and it displayed maximal activity at 85 degrees C and pH 8.0 with a turnover number of 376 s-1. The enzyme showed classical saturation kinetics and no sigmoidicity was detected at different pH values and temperatures. At 60 degrees C and in the presence of 0.1 mM MnCl2, the Michaelis constants for malate, NADP, and NAD were 18, 3, and 250 microM, respectively. The S. solfataricus malic enzyme was shown to be very thermostable.     

Functional roles of ATP-binding residues in the catalytic site of human mitochondrial NAD(P)+-dependent malic enzyme

Human mitochondrial NAD(P)+-dependent malic enzyme is inhibited by ATP. The X-ray crystal structures have revealed that two ATP molecules occupy both the active and exo site of the enzyme, suggesting that ATP might act as an allosteric inhibitor of the enzyme. However, mutagenesis studies and kinetic evidences indicated that the catalytic activity of the enzyme is inhibited by ATP through a competitive inhibition mechanism in the active site and not in the exo site. Three amino acid residues, Arg165, Asn259, and Glu314, which are hydrogen-bonded with NAD+ or ATP, are chosen to characterize their possible roles on the inhibitory effect of ATP for the enzyme. Our kinetic data clearly demonstrate that Arg165 is essential for catalysis. The R165A enzyme had very low enzyme activity, and it was only slightly inhibited by ATP and not activated by fumarate. The values of K(m,NAD) and K(i,ATP) to both NAD+ and malate were elevated. Elimination of the guanidino side chain of R165 made the enzyme defective on the binding of NAD+ and ATP, and it caused the charge imbalance in the active site. These effects possibly caused the enzyme to malfunction on its catalytic power. The N259A enzyme was less inhibited by ATP but could be fully activated by fumarate at a similar extent compared with the wild-type enzyme. For the N259A enzyme, the value of K(i,ATP) to NAD+ but not to malate was elevated, indicating that the hydrogen bonding between ATP and the amide side chain of this residue is important for the binding stability of ATP. Removal of this side chain did not cause any harmful effect on the fumarate-induced activation of the enzyme. The E314A enzyme, however, was severely inhibited by ATP and only slightly activated by fumarate. The values of K(m,malate), K(m,NAD), and K(i,ATP) to both NAD+ and malate for E314A were reduced to about 2-7-folds compared with those of the wild-type enzyme. It can be concluded that mutation of Glu314 to Ala eliminated the repulsive effects between Glu314 and malate, NAD+, or ATP, and thus the binding affinities of malate, NAD+, and ATP in the active site of the enzyme were enhanced.     

Purification and properties of the NAD(P)-dependent malic enzyme from human placental mitochondria

The NAD(P)-dependent malic enzyme from human term placental mitochondria was purified 108-fold with a final yield of 72% and specific activity of about 2 mumol per minute per milligram protein. The final preparation was completely free of fumarase, malic, and lactic dehydrogenases. Divalent cations were required for NAD(P)-dependent malic enzyme activity, Mn2+ and Co2+ were by far more effective activators than Mg2+ and Ni2+, whereas the reaction did not proceed in the presence of Ca2+. The optimum pH with NAD and NADP as coenzymes was at around 7.1 and 6.4, respectively. The ratio of the rate of NAD:NADP reduction was 7.4 and 1.3 at pH 7.1 and 6.4, respectively. The enzyme is activated by succinate and fumarate and inhibited by ATP. In the absence of fumarate the Michaelis constants for L-malate and NAD were 2.82 and 0.33 mM; and in the presence of fumarate 1.18 and 0.22 mM, respectively. This study presents the first report showing the purification and kinetic properties of NAD(P)-dependent malic enzyme from human tissue.     

Ascaris suum NAD-malic enzyme is activated by L-malate and fumarate binding to separate allosteric sites

The kinetic mechanism of activation of the mitochondrial NAD-malic enzyme from the parasitic roundworm Ascaris suum has been studied using a steady-state kinetic approach. The following conclusions are suggested. First, malate and fumarate increase the activity of the enzyme in both reaction directions as a result of binding to separate allosteric sites, i.e., sites that exist in addition to the active site. The binding of malate and fumarate is synergistic with the K(act) decreasing by >or=10-fold at saturating concentrations of the other activator. Second, the presence of the activators decreases the K(m) for pyruvate 3-4-fold, and the K(i) (Mn) >or=20-fold in the direction of reductive carboxylation; similar effects are obtained with fumarate in the direction of oxidative decarboxylation. The greatest effect of the activators is thus expressed at low reactant concentrations, i.e., physiologic concentrations of reactant, where activation of >or=15-fold is observed. A recent crystallographic structure of the human mitochondrial NAD malic enzyme [13] shows fumarate bound to an allosteric site. Site-directed mutagenesis was used to change R105, homologous to R91 in the fumarate activator site of the human enzyme, to alanine. The R105A mutant enzyme exhibits the same maximum rate and V/K(NAD) as does the wild-type enzyme, but 7-8-fold decrease in both V/K(malate) and V/K(Mg), indicating the importance of this residue in the activator site. In addition, neither fumarate nor malate activates the enzyme in either reaction direction. Finally, a change in K143 (a residue in a positive pocket adjacent to that which contains R105), to alanine results in an increase in the K(act) for malate by about an order of magnitude such that it is now of the same magnitude as the K(m) for malate. The K143A mutant enzyme also exhibits an increase in the K(act) for fumarate (in the absence of malate) from 200 microM to about 25 mM.     

Mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate.

The mechanism of activation of the NAD-malic enzyme from Ascaris suum by fumarate has been probed using initial velocity studies, deuterium isotope effects, and isotope partitioning of the E:Mg:malate complex. Fumarate exerts its activating effect by decreasing the off-rate for malate from the E:Mg:malate and E:NAD:Mg:malate complexes. Fumarate is a positive heterotropic effector of the NAD-malic enzyme at low concentrations (K act approximately 0.05 mM) and an inhibitor competitive against malate (Ki approximately 25 mM). The activation by fumarate results in a decrease in the Ki malate and an increase in V/K malate of about 2-fold, while the maximum velocity remains constant. Isotope partitioning studies of E:Mg:[14C]malate indicate that the presence of fumarate results in a decrease in the malate off-rate constant by about 2.2-fold. The deuterium isotope effects on V and V/K malate are both 1.6 +/- 0.1 in the absence of fumarate, while in the presence of 0.5 mM fumarate DV is 1.6 +/- 0.1 and D(V/K malate) is 1.1 +/- 0.1. These data are also consistent with a decrease in the off-rate for malate from E:NAD:Mg:malate, resulting in an increase in the forward commitment factor for malate and manifested as a lower value for D(V/K malate). There is a discrimination between active and activator sites for the binding of dicarboxylic acids, with the activator site preferring the extended configuration of 4-carbon dicarboxylic acids, while the active site prefers a configuration in which the 4-carboxyl is twisted out of the C1-C3 plane. The physiologic importance and regulatory properties of fumarate in the parasite are also discussed.     

AMP deaminase from yeast. Role in AMP degradation, large scale purification, and properties of the native and proteolyzed enzyme

Eukaryotes have been proposed to depend on AMP deaminase as a primary step in the regulation of intracellular adenine nucleotide pools. This report describes 1) the role of AMP deaminase in adenylate metabolism in yeast cell extracts, 2) a method for large scale purification of the enzyme, 3) the kinetic properties of native and proteolyzed enzymes, 4) the kinetic reaction mechanism, and 5) regulatory interactions with ATP, GTP, MgATP, ADP, and PO4. Allosteric regulation of yeast AMP deaminase is of physiological significance, since expression of the gene is constitutive (Meyer, S. L., Kvalnes-Krick, K. L., and Schramm, V. L. (1989) Biochemistry 28, 8734-8743). The metabolism of ATP in cell-free extracts of yeast demonstrates that AMP deaminase is the sole pathway of AMP catabolism in these extracts. Purification of the enzyme from bakers' yeast yields a proteolytically cleaved enzyme, Mr 86,000, which is missing 192 amino acids from the N-terminal region. Extracts of Escherichia coli containing a plasmid with the gene for yeast AMP deaminase contained only the unproteolyzed enzyme, Mr 100,000. The unproteolyzed enzyme is highly unstable during purification. Substrate saturation plots for proteolyzed AMP deaminase are sigmoidal. In the presence of ATP, the allosteric activator, the enzyme exhibits normal saturation kinetics. ATP activates the proteolyzed AMP deaminase by increasing the affinity for AMP from 1.3 to 0.2 mM without affecting VM. Activation by ATP is more efficient than MgATP, with half-maximum activation constants of 6 and 80 microM, respectively. The kinetic properties of the proteolyzed and unproteolyzed AMP deaminase are similar. Thus, the N-terminal region is not required for catalysis or allosteric activation. AMP deaminase is competitively inhibited by GTP and PO4 with respect to AMP. The inhibition constants for these inhibitors decrease in the presence of ATP. ATP, therefore, tightens the binding of GTP, PO4, and AMP. The products of the reaction, NH3 and IMP, are competitive inhibitors against substrate, consistent with a rapid equilibrium random kinetic mechanism. Kinetic dissociation constants are reported for the binary and ternary substrate and product complexes and the allosteric modulators.     

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.     

Purification and properties of mitochondrial malate dehydrogenase from unfertilized eggs of the sea urchin, Anthocidaris crassispina

Purification and characterization of mitochondrial malate dehydrogenase [EC 1.1.1.37] from unfertilized eggs of the sea urchin, Anthocidaris crassispina, are described. The purification method consisted of dextran sulfate fractionation, Blue Dextran Sepharose chromatography, Phenyl-Sepharose hydrophobic chromatography and DEAE-cellulose chromatography. The enzyme was purified 771-fold with a 7% yield from the crude extract. The purified enzyme appeared homogeneous on polyacrylamide gel electrophoresis under both native and denatured conditions. After incubation at 45 degrees C for 50 min, the enzyme lost about 90% of its activity. In the presence of NADH, however, the enzyme was protected against the heat denaturation. The native enzyme had a molecular weight of about 65,000 and probably consisted of two identical subunits. In the reduction of oxaloacetate with NADH, a broad optimum pH ranging from 8.2 to 9.4 was found with 50 mM Tris-HCl and glycine-NaOH buffers. Sodium phosphate buffer apparently activated the enzyme. The apparent Km values for oxaloacetate and NADH were 19 microM and 30 microM, respectively. The optimum pH for malate oxidation with NAD+ was 10.2 in 50 mM NaHCO3-Na2CO3 buffer. The apparent Km values for malate and NAD+ were 7.0 mM and 0.6 mM, respectively. Zinc ion, sulfite ion, p-chloromercuriphenylsulfonate and adenine nucleotides strongly inhibited the enzyme.     

Molecular mechanism for the regulation of human mitochondrial NAD(P)+-dependent malic enzyme by ATP and fumarate

The regulation of human mitochondrial NAD(P)+-dependent malic enzyme (m-NAD-ME) by ATP and fumarate may be crucial for the metabolism of glutamine for energy production in rapidly proliferating tissues and tumors. Here we report the crystal structure at 2.2 A resolution of m-NAD-ME in complex with ATP, Mn2+, tartronate, and fumarate. Our structural, kinetic, and mutagenesis studies reveal unexpectedly that ATP is an active-site inhibitor of the enzyme, despite the presence of an exo binding site. The structure also reveals the allosteric binding site for fumarate in the dimer interface. Mutations in this binding site abolished the activating effects of fumarate. Comparison to the structure in the absence of fumarate indicates a possible molecular mechanism for the allosteric function of this compound.     

Purification of particulate malate dehydrogenase and phosphoenolpyruvate carboxykinase from Leishmania mexicana mexicana

The particulate activities of Leishmania mexicana mexicana amastigote malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37) and phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating) EC 4.1.1.49) have been purified to apparent electrophoretic homogeneity by hydrophobic interaction chromatography using Phenyl-Sepharose CL-4B, affinity chromatography using 5'AMP-Sepharose 4B, and gel filtration using Sephadex G-100. Malate dehydrogenase was purified 150-fold overall with a final specific activity of 1230 units/mg protein and a recovery of 63%. Phosphoenolpyruvate carboxykinase was purified 132-fold with a final specific activity of 30.3 units/mg protein and a recovery of 20%. Molecular weights determined by gel filtration and SDS-gel electrophoresis were 39 800 and 33 300 for malate dehydrogenase and 63 100 and 65 100 for phosphoenolpyruvate carboxykinase, respectively. Kinetic studies with malate dehydrogenase assayed in the direction of oxaloacetic acid reduction showed a Km(NADH) of 41 microM and a Km(oxaloacetic acid) of 39 microM. For malate oxidation there was a Km(malate) of 3.6 mM and a Km(NAD) of 0.79 mM. Oxaloacetic acid exhibited substrate inhibition at concentrations greater than 0.83 mM and malate was found to be a product inhibitor at high concentrations. However, there was no modification of enzyme activity by a number of glycolytic intermediates and cofactors, suggesting that malate dehydrogenase is not a major regulatory enzyme in L. m. mexicana. The results show that these L. m. mexicana amastigote enzymes are in several ways similar to their mammalian counterparts; nevertheless, their apparent importance and unique subcellular organization in the parasite make them potential targets for chemotherapeutic attack.     

Inhibition by hydroxymalonate of malate dependent biosynthesis of progesterone in the mitochondrial fraction of human term placenta

It has been shown that the conversion of cholesterol to progesterone by human term placental mitochondria incubated in the presence of malate or fumarate was inhibited by hydroxymalonate—an inhibitor of malic enzyme. No inhibition was observed when mitochondria were incubated in the presence of citrate or isocitrate. The degree of inhibition by hydroxymalonate of partly purified NAD(P)-linked malic enzyme activity was identical to that of both malate dependent pyruvate and progesterone formation by intact mitochondria. These data strongly support a previous suggestion that malic enzyme plays an important role in the malate dependent progesterone biosynthesis by human placental mitochondria.     

Arginyl-tRNA synthetase from Escherichia coli, purification by affinity chromatography, properties, and steady-state kinetics

A Blue Sephadex G-150 affinity column adsorbs the arginyl-tRNA synthetase of Escherichia coli K12 and purifies it with high efficiency. The relatively low enzyme content was conveniently purified by DEAE-cellulose chromatography, affinity chromatography, and fast protein liquid chromatography to a preparation with high activity capable of catalyzing the esterification of about 23,000 nmol of arginine to the cognate tRNA per milligram of enzyme within 1 min, at 37 degrees C, pH 7.4. The turnover number is about 27 s-1. The purification was about 1200-fold, and the overall yield was more than 30%. The enzyme has a single polypeptide chain of about Mr 70,000 and binds arginine and tRNA with 1:1 stoichiometry. For the aminoacylation reaction, the Km values at pH 7.4, 37 degrees C, for various substrates were determined: 12 microM, 0.9 mM, and 2.5 microM for arginine, ATP, and tRNA, respectively. The Km value for cognate tRNA is higher than those of most of the aminoacyl-tRNA synthetase systems so far reported. The ATP-PPi exchange reaction proceeds only in the presence of arginine-specific tRNA. The Km values of the exchange at pH 7.2, 37 degrees C, are 0.11 mM, 2.9 mM, and 0.5 mM for arginine, ATP, and PPi, respectively, with a turnover number of 40 s-1. The pH dependence shows that the reaction is favored toward slightly acidic conditions where the aminoacylation is relatively depressed.     

Binary and ternary complexes of malate dehydrogenase with substrates and substrate analogs

Hydroxypyrenetrisulfonate binds to pig mitochondrial malate dehydrogenase (L-malate: NAD+ oxidoreductase, EC 1.1.1.37) in the presence and absence of coenzymes with a stoichiometry of one dye molecule/enzyme subunit. Binding is competitive with substrates and known substrate analogs as well as with squaric acid, a newly detected analog forming a ternary complex with enzyme/NAD+ similar to enzyme/NAD+/sulfite. Displacement of hydroxypyrenetrisulfonate by substrates and analogs was used to determine dissociation constants of binary and ternary complexes. Binary complexes form with dissociation constants of about 10 mM. They may be important for kinetic studies at high substrate concentrations where oxaloacetate inhibition and malate activation have been described.     

Influential factor contributing to the isoform-specific inhibition by ATP of human mitochondrial NAD(P)+-dependent malic enzyme: functional roles of the nucleotide binding site Lys346

Human mitochondrial NAD(P)(+)-dependent malic enzyme (m-NAD-ME) is a malic enzyme isoform with dual cofactor specificity, ATP inhibition and substrate cooperativity. The determinant of ATP inhibition in malic enzyme isoforms has not yet been identified. Sequence alignment of nucleotide-binding sites of ME isoforms revealed that Lys346 is conserved uniquely in m-NAD-ME. In other ME isoforms, this residue is serine. As the inhibitory effect of ATP is more pronounced on m-NAD-ME than on other ME isoforms, we have examined the possible role of Lys346 by replacing it to alanine, serine or arginine. Our kinetic data indicate that the K346S mutant enzyme displays a shift in its cofactor preference from NAD(+) to NADP(+) upon increasing k(cat,NADP) and decreasing K(m,NADP). Furthermore, the cooperative binding of malate becomes less significant in human m-NAD-ME after mutation of Lys346. The h value for the wild-type is close to 2, but those of the K346 mutants are approximately 1.5. The K346 mutants can also be activated by fumarate and the cooperative effect can be abolished by fumarate, suggesting that the allosteric property is retained in these mutants. Our data strongly suggest that Lys346 in human m-NAD-ME is required for ATP inhibition. Mutation of Lys346 to Ser or Ala causes the enzyme to be much less sensitive to ATP, similar to cytosolic NADP-dependent malic enzyme. Substitution of Lys to Arg did not change the isoform-specific inhibition of the enzyme by ATP. The inhibition constants of ATP are increased for K346S and K346A, but are similar to those of the wild-type for K346R, suggesting that the positive charge rather than group specificity is required for binding affinity of ATP. Thus, ATP inhibition is proposed to be determined by the electrostatic potential involving the positive charge on the side chain of Lys346.     

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.     

Differential fumarate binding to Arabidopsis NAD+-malic enzymes 1 and -2 produces an opposite activity modulation

Arabidopsis mitochondria contain two NAD(+)-malic enzymes, NAD-ME1 and NAD-ME2. These proteins have similar affinity for their substrates but display opposite regulation by fumarate, which strongly stimulates NAD-ME1 but inhibits NAD-ME2 activity. Here, the interaction of NAD-ME1 and -2 with fumarate was investigated by kinetic approaches, urea denaturation assays and intrinsic fluorescence quenching, in the absence and presence of NAD(+). Fumarate inhibited NAD-ME2 at saturating, but not at low, levels of NAD(+), and it behaved as competitive inhibitor with respect to L-malate. In contrast, NAD-ME1 fumarate activation was higher at suboptimal NAD(+) concentrations. In the absence of cofactor, the fluorescence of both NAD-ME1 and -2 is quenched by fumarate. However, for NAD-ME2 the quenching arises from a collisional phenomenon, while in NAD-ME1 the fluorescence decay can be explained by a static process that involves fumarate binding to the protein. Furthermore, the residue Arg84 of NAD-ME1 is essential for fumarate binding, as the mutant protein R84A exhibits a collisional quenching by this metabolite. Together, the results indicate that the differential fumarate regulation of Arabidopsis NAD-MEs, which is further modulated by NAD(+) availability, is related to the gaining of an allosteric site for fumarate in NAD-ME1 and an active site-associated inhibition by this C(4)-organic acid in NAD-ME2.     

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.     

The Respiratory Chain of Plant Mitochondria: XI. Electron Transport from Succinate to Endogenous Pyridine Nucleotide in Mung Bean Mitochondria

Energy-linked reverse electron transport from succinate to endogenous NAD in tightly coupled mung bean (Phaseolus aureus) mitochondria may be driven by ATP if the two terminal oxidases of these mitochondria are inhibited, or may be driven by the free energy of succinate oxidation. This reaction is specific to the first site of energy conservation of the respiratory chain; it does not occur in the presence of uncoupler. If mung bean mitochondria become anaerobic during oxidation of succinate, their endogenous NAD becomes reduced in the presence of uncoupler, provided that both inorganic phosphate (P_i) and ATP are present. No reduction occurs in the absence of P_i, even in the presence of ATP added to provide a high phosphate potential. If fluorooxaloacetate is present in the uncoupled, aerobic steady state, no reduction of endogenous NAD occurs on anaerobiosis; this compound is an inhibitor of malate dehydrogenase. This result implies that endogenous NAD is reduced by malate formed from the fumarate generated during succinate oxidation. The source of free energy is most probably the endogenous energy stores in the form of acetyl CoA, or intermediates convertible to acetyl CoA, which removes the oxaloacetate formed from malate, thus driving the reaction towards reduction of NAD.     

Phosphofructokinase from muscle of the marine giant barnacle Austromegabalanus psittacus: kinetic characterization and effect of in vitro phosphorylation

The kinetic properties of phosphofructokinase from muscle of the giant cirripede Austromegabalanus psittacus were characterized, after partial purification by ion exchange chromatography on DEAE-cellulose. This enzyme showed differences regarding PFKs from other marine invertebrates: the affinity for fructose 6-phosphate (Fru 6-P) was very low, with an S(0.5) of 22.6+/-1.4 mM (mean+/-S.D., n=3), and a high cooperativity (n(H) of 2.90+/-0.21; mean+/-S.D., n=3). The barnacle PFK showed hyperbolic saturation kinetics for ATP (apparent K(m ATP)=70 microM, at 5 mM Fru 6-P, in the presence of 2 mM ammonium sulfate). ATP concentrations higher than 1 mM inhibited the enzyme. Ammonium sulfate activated the PFK several folds, increasing the affinity of the enzyme for Fru 6-P and V(max). 5'-AMP (0.2 mM) increased the affinity for Fru 6-P (S(0.5) of 6.2 mM). Fructose 2,6-bisphosphate activated the PFK, with a maximal activation at concentrations higher than 2 microM. Citrate reverted the activation of PFK produced by 0.2 mM 5'-AMP (IC(50 citrate)=2.0 mM), producing a higher inhibition than that exerted on other invertebrate PFKs. Barnacle muscular PFK was activated in vitro after exposure to exogenous cyclic-AMP (0.1 mM) as well as by phosphatidylserine (50 microg/ml), indicating a possible control by protein kinase A and a phospholipid dependent protein kinase (PKC). The results suggest a highly regulated enzyme in vivo, by allosteric mechanisms and also by protein phosphorylation.