Purification and properties of malic dehydrogenase from Trichomonas gallinae

The malic dehydrogenase (MDH², l-malate: NAD oxidoreductase, E.C. 1,1.1.37) of Trichomonas gallinae was purified 215-fold and characterized. The molecular weight was found to be 72,000 and the enzyme protein contained essential cations and sulfhydryl groups. Polyacrylamide gel electrophoresis before and after extensive purification yielded a single band of malic dehydrogenase activity strongly suggesting only one molecular form of the enzyme. Analysis of kinetic data yielded the following K_m values: oxalocetate, 16 μM; malate, 200 μM; NADH 11 μM; and NAD, 70 μM. The enzyme was absolutely specific for l-malic acid, NAD, and NADH. The enzyme exhibited a broad band of heat stability with an optimum of 51 C. The pH optimum in the direction of oxalacetate reduction was 9.0. The pH optima in the reverse direction were 9.0 and 10.5 A role for this enzyme in T. gallinae metabolism is discussed.     

Allosteric and isosteric modifiers of NADH binding to cytoplasmic malic dehydrogenase

The binding of NADH to cytoplasmic malic dehydrogenase is shown to be affected by a number of added ligands. One class of ligands appear to be analogs of a substrate for the enzyme, $\text{L}$-malate. These alter the binding constant for NADH without affecting the cooperativity of binding. In contrast, fructose-1,6-diphosphate behaves as an allosteric inhibitor at low enzyme concentrations, apparently by shifting the monomer-dimer equilibrium of the protein to the cooperatively binding dimer. The significance of these results are discussed in terms of a proposed regulatory function for the enzyme.     

Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.     

Stereospecific effects of ascorbic acid and analogues on D1 and D2 agonist binding.

Ascorbic acid inhibited the specific binding of both the D1 agonist, [3H] SKF 38393, and the D2 agonist, [3H] N-0437 at physiologically relevant concentrations. This inhibition was both stereospecific and receptor selective. Using ligand concentrations approximating their KD's, the IC50's for ascorbate and two structural analogues, isoascorbate and D-glucoascorbate, were determined. The rank order of IC50's at both D1 and D2 were D-glucoascorbate greater than isoascorbate greater than ascorbate. However, the IC50 for each compound was greater at D1 than D2. Evaluation of the relationship between the IC50 for ascorbate and the ligand concentration using both the D1 and the D2 ligand yielded data inconsistent with competitive inhibition models. Preliminary experiments were conducted to evaluate the site and type of inhibition with results consistent with an allostearic effect at the level of the receptor.     

Mitochondrial malate dehydrogenase and malic enzyme of a filarial worm Setaria digitata: some properties and effects of drugs and herbal extracts.

Mitochondrial malate dehydrogenase (mMDH) and malic enzyme (mME) of a filarial worm Setaria digitata were studied. mMDH exhibited the highest activities in the oxidation and reduction reactions at pH 9.5 and pH 6.2, respectively, while mME did so in the malate decarboxylation reaction at pH 6.8. mME showed no detectable activity on the pyruvate carboxylation direction. The Km values for malate (1.7 mM) and oxaloacetate (0.17 mM) and the ratio of Vmax oxidation: Vmax reduction (2.73) tend to favor the oxaloacetate reduction by mMDH. mME showed a relatively high Km value of 8.3 mM, for malate decarboxylation. A drug, diethylcarbamazine citrate (DEC-C), did not change appreciably the activity of either mMDH or mME, while filarin (a drug of herbal origin) effectively inhibited mMDH. The leaf extracts of Ocimum sanctum, Lawsonia inermis and Calotropis gigantea and leaf and flower extracts of Azadirachta indica were, however, found to inhibit both mMDH and mME.     

Studies on the catalytic mechanism of H2-forming methylenetetrahydromethanopterin dehydrogenase: para-ortho H2 conversion rates in H2O and D2O

H₂–forming N ⁵,N ¹⁰ –methylenetetrahydromethanopterin dehydrogenase is a novel type of hydrogenase that contains neither nickel nor iron-sulfur clusters. Evidence has been presented that the reaction mechanism catalyzed by the enzyme is very similar to that of the formation of carbocations and H₂ from alkanes under superacidic conditions. We present here further results in support of this mechanism. It was found that the purified enzyme per se did not catalyze the conversion of para H₂ to ortho H₂, a reaction catalyzed by all other hydrogenases known to date. However, it catalyzed the conversion in the presence of the substrate N ⁵,N ¹⁰ –methenyltetrahydromethanopterin (CH≡H₄MPT⁺), indicating that for heterolytic cleavage of H₂ the enzyme-CH≡H₄MPT⁺ complex is required. In D₂O, the formation of HD and D₂ from H₂ rather than a para–ortho H₂ conversion was observed, indicating that after heterolytic cleavage of H₂ the dissociation of the proton from the enzyme-substrate complex is fast relative to the re-formation of free H₂.     

Isotope effects on binding of NAD+ to lactate dehydrogenase

The isotope effect on binding [4-2H]NAD+ and [4-3H]NAD+ to lactate dehydrogenase has been shown to be 1.10 +/- 0.03 by whole molecule isotope ratio mass spectrometry and 1.085 +/- 0.01 by 3H/14C scintillation counting. These values demonstrate that specific interactions of the nicotinamide ring with the enzyme make the C-H bond at C-4 less stiff in the binary complex.     

Purification and properties of the NAD+-dependent (type D) and O2-dependent (type O) forms of rat liver xanthine dehydrogenase.

The xanthine-oxidizing enzyme of rat liver has been purified as an NAD⁺-dependent dehydrogenase (type D) and as the O₂-dependent oxidase (type O). The purified D and O variants are nearly homogenous as judged by polyacrylamide discontinuous gel electrophoresis and are indistinguishable on sodium dodecyl sulfate-urea gels. The absorption spectrum of the type D enzyme is indistinguishable from that of the type O enzyme and closely resembles the spectra of xanthine-oxidizing enzymes from other sources. The types D and O enzymes have essentially the same cofactor composition. Oxidation of xanthine by type D is stimulated by NAD⁺ with concomitant NADH formation. Type D is able to utilize NADH as well as xanthine as electron donor to various acceptors, in contrast to type O that is unable to oxidize NADH. Arsenite, cyanide and methanol completely abolish xanthine oxidation by the type D enzyme while affecting the activities with NADH to varying extents. In these respects rat liver xanthine dehydrogenase closely resembles chicken liver xanthine dehydrogenase. However, in contrast to the avian enzyme, the purified rat liver enzyme is unstable as a dehydrogenase and is gradually converted to an oxidase. This conversion is accompanied by an increase in the aerobic xanthine → cytochrome c activity. The native type D enzyme in rat liver extracts is precipitable with antibody prepared against purified type O. The K_m for xanthine is not significantly different for the two forms.     

The effect of salinity on the malic dehydrogenase of pea roots

Effect of salinity on malate dehydrogenase activity was studied.     

Biological effects of D2O administration to Coturnix japonica

Levels of 7.8, 18.5 and 26 mole % deuterium oxide were administered sequentially to $\text{Coturnix japonica}$ (Japanese quail) via the drinking water. The primary effect observed was on egg frequency, which decreased from a normal level of 0.89 for 7.8 mole % D₂O to a low of 0.38 during the administration of 26 mole % D₂O. Adverse symptoms, such as hyperexcitability, convulsions, skin ulcerations, comatosity, weight loss, or death, which have been associated with deuterium toxicity in other animals, were not observed in these experiments. The amount of deuterium deposited in the water of the egg was 6.9, 13.98, and 19.83 mole % when 7.8, 18.5 and 26 mole % deuterium respectively was administered. For each period, the deuterium content of egg water rapidly reached a maximum concentration after which the concentration decreased slightly. This dilution effect has not been noted previously in body fluids from other animals.