Purification and properties of mitochondrial malate dehydrogenase of Toxocara canis muscle

Mitochondrial malate dehydrogenase was purified from muscle extracts of Toxocara canis by means of Sephadex G-100 gel filtration, DEAE-Sephadex ion-exchange chromatography and 5'AMP-Sepharose 4B affinity chromatography. The purified enzyme showed an optimum pH for the reduction of oxaloacetate of 7.3 in Tris-HCl buffer and of pH 7.5-7.8 in phosphate buffer. The m-MDH showed values of 3.2 kcal/mol and 10.5 kcal/mol for the energy of activation, calculated from the Arrhenius equation. The mitochondrial enzyme was found to be more susceptible to thermal inactivation as compared with the cytosolic isoenzyme. Kinetic experiments showed that the m-MDH of Toxocara canis is inhibited by excess oxaloacetate but not by excess NADH. The apparent Km for oxaloacetate reduction was 53 microM and 0.54 mM for L-malate oxidation.     

Effects of temperature on the mitochondrial malate dehydrogenase of adult muscle of Toxocara canis

Purified mitochondrial malate dehydrogenase isoenzyme (m-MDH) of Toxocara canis muscle presented maximum activity at 48 degrees C. A clear change in slope of the Arrhenius plot was observed. The energy of activation calculated for the catalytic process showed values of 3.2 kcal/mol and 10.5 kcal/mol. Thermal inactivation of m-MDH showed that it is more thermolabile than the s-isoenzyme. The inactivation of the enzyme by heat could be reduced at least in part by the addition of 0.1 mM NADH. The heat denaturation showed to be a first-order process. The rate constant (k) was calculated as being of the order of 5.28 X 10(-4) s-1 at 40 degrees C. The activation energy for the heat inactivation process was 16.45 kcal/mol between 30 degrees C and 40 degrees C and 13.79 kcal/mol between 40 degrees C and 48 degrees C.     

The mitochondrial genome of Toxocara canis

Toxocara canis (Ascaridida: Nematoda), which parasitizes (at the adult stage) the small intestine of canids, can be transmitted to a range of other mammals, including humans, and can cause the disease toxocariasis. Despite its significance as a pathogen, the genetics, epidemiology and biology of this parasite remain poorly understood. In addition, the zoonotic potential of related species of Toxocara, such as T. cati and T. malaysiensis, is not well known. Mitochondrial DNA is known to provide genetic markers for investigations in these areas, but complete mitochondrial genomic data have been lacking for T. canis and its congeners. In the present study, the mitochondrial genome of T. canis was amplified by long-range polymerase chain reaction (long PCR) and sequenced using a primer-walking strategy. This circular mitochondrial genome was 14162 bp and contained 12 protein-coding, 22 transfer RNA, and 2 ribosomal RNA genes consistent for secementean nematodes, including Ascaris suum and Anisakis simplex (Ascaridida). The mitochondrial genome of T. canis provides genetic markers for studies into the systematics, population genetics and epidemiology of this zoonotic parasite and its congeners. Such markers can now be used in prospecting for cryptic species and for exploring host specificity and zoonotic potential, thus underpinning the prevention and control of toxocariasis in humans and other hosts.     

Dissociation of mitochondrial malate dehydrogenase

The kinetics of the dissociation reaction under acidic conditions of the dimeric pig and chicken mitochondrial malate dehydrogenases (EC 1.1.1.37) have been studied. The dissociation of the pig enzyme is completely reversible. The pK for dissociation determined by light-scattering measurements agrees within experimental error with the pK value of 5.25 measured for a tyrosine-carboxylate pair. The rate constants for the dissociation reaction and for the protonation process of this tyrosine are in close agreement. Thus, the tyrosine-carboxylate pair can be used as indicator of the dissociation reaction. The dissociation of the chicken enzyme proceeds around pH 4.5 at a much lower rate. A true equilibrium between dimer and monomers is not found, since the monomer gradually unfolds at this pH. The monomers of both enzymes, pig and chicken mitochondrial malate dehydrogenase, show the same stability towards acid. The difference in stability of the dimeric forms, therefore, must be due to an altered subunit contact area.     

Characterization of a cytosolic malate dehydrogenase cDNA which encodes an isozyme toward oxaloacetate reduction in wheat

Malate dehydrogenase (MDH), which is ubiquitous in nature, catalyzes the interconversion of oxaloacetate and malate. Higher plants contain multiple forms of MDH that differ in co-enzyme specificity, subcellular localization and physiological function. Cytosolic NAD-dependent MDH (cyMDH) is one class of MDH that has not been extensively characterized in plants. Here we report the cloning of a cDNA from wheat by RT-PCR and cDNA library screening, which is designated as TaMDH. Sequence analysis indicated that TaMDH exhibits a highly similarity to other plant cyMDHs. Immunological analysis confirmed that TaMDH encoded a cytosolic NAD-dependent MDH. The secondary and three-dimensional structures of TaMDH were analyzed by molecular modeling. DNA gel-blot analyses demonstrated that TaMDH gene exists as two copies in the wheat genome. RNA and protein gel-blot hybridization indicated that both TaMDH mRNA and protein were constitutively expressed in vegetative tissues of wheat, with slightly lower levels in roots than in leaves and stems. In silico analysis indicated that TaMDH was also expressed in various reproductive tissues and tissues under many different stress conditions. Kinetic analysis of bacterially expressed and purified protein confirmed that TaMDH catalyzed a reaction driven towards malate synthesis, which is consistent with other cyMDHs. Evolutionary analysis showed that this class of genes evolved from a very ancestral gene. The cyMDH represents an ancestral form of MDH, which is highly conserved in plants, animals and bacteria. This implies that cyMDHs are housekeeping genes and may have very essential functions in plant metabolism.     

Kinetics of alpha-dicarbonyls reduction by L-glycol dehydrogenase from hen muscle

Michaelis constants of L-glycol dehydrogenase from hen muscle (isozyme of pI 7.2) for the alpha-dicarbonyls tested (glyoxal, 2,3-pentanedione, methylglyoxal, and diacetyl) range from 35 microM for pentanedione to 0.41 mM for glyoxal. The enzyme shows a high affinity for NADPH, Km (2.2-3.1 microM), and Ks (1.2-1.9 microM) being so much lower than its tissue concentration that L-glycol dehydrogenase has to operate in vivo saturated with the coenzyme; this condition is very unfavorable to play a role in regulating the equilibrium oxidized/reduced forms of the pyridine nucleotides, as it has been proposed for some similar enzymes. Convergence of the double reciprocal plots and the pattern of inhibition by products and by acetone, a substrate analog, demonstrate that glyoxal reduction--and most likely that of diacetyl--proceeds via an ordered Bi-Bi mechanism in which NADPH is fixed before the addition of the carbonyl; the reduction of methylglyoxal and 2,3-pentanedione could follow the same model, but our experimental results are also consistent with that of Theorell-Chance.     

Subunit dissociation of mitochondrial malate dehydrogenase.

Fluorescence polarization studies of porcine mitochondrial malate dehydrogenase labeled with fluorescein isothiocyanate or fluorescamine indicated a concentration-dependent dissociation of the dimeric molecule with a KD OF 2 X 10(7) N at pH 8.0. These results were confirmed by the concentration dependence of the stability of the enzyme at elevated temperatures and the creation of hybrid molecules with fluorescein and Rhodamine B labeled subunits, in which energy transfer was observed. The binding of NADH resulted in a small shift of the subunit dissociation curve toward monomer, demonstrating that monomer has twice the affinity for reduced coenzyme. NAD+ binding prevented dissociation of the dimer, even at concentrations below 10(-8) N. These results indicate that binding of reduced or oxidized coenzymes results in different conformation changes, which are transferred to the subunit interface.     

Lipoic acid inhibition of mitochondrial malate dehydrogenase

Porcine heart mitochondrial malate dehydrogenase (L-malate:NAD⁺ oxidoreductase, EC 1.1.1.37) has been shown to be inhibited by extremely low concentrations of lipoic acid. The actual inhibitor was found to be a high molecular weight substance, which can be separated by gel permeation from the non-inhibitory monomeric form of lipoic acid. This inhibitor has been identified as a polymeric form of lipoic acid.     

Aggregation states of mitochondrial malate dehydrogenase.

The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 +/- 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution of a mMDH-fluorescein conjugate, prepared using a Tris buffer protocol, did show dissociation, which can be attributed to aggregates present in these preparations. These results are considered in light of the disparities in the literature concerning the properties of the mMDH dimer-monomer equilibrium.     

Kinetics of the coupled reaction catalysed by a fusion protein of yeast mitochondrial malate dehydrogenase and citrate synthase.

The mechanistic implications of the kinetic behaviour of a fusion protein of mitochondrial malate dehydrogenase and citrate synthase have been reanalysed in view of predictions based on experimentally determined kinetic parameter values for the dehydrogenase and synthase activities of the protein. The results show that the time-course of citrate formation from malate in the coupled reaction catalysed by the fusion protein can be most satisfactorily accounted for in terms of a free-diffusion mechanism when consideration is taken to the inhibitory effects of NADH and oxaloacetate on the malate dehydrogenase activity. The effect of aspartate aminotransferase on the coupled reaction is likewise fully consistent with that expected for a free-diffusion mechanism. It is concluded that no tenable kinetic evidence is available to support the proposal that the fusion protein catalyses citrate formation from malate by a mechanism involving channelling of the intermediate oxaloacetate.     

The reduction of aromatic alpha-keto acids by cytoplasmic malate dehydrogenase and lactate dehydrogenase

This study demonstrates that cytoplasmic malate dehydrogenase (MDH-s) catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, that the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; EC 1.1.1.96) is identical to MDH-s, and that the reduction of aromatic alpha-keto acids is due predominantly to a previously unrecognized secondary activity of MDH-s and the remainder is due to the previously recognized activity of lactate dehydrogenase (LDH) toward aromatic keto-acids. MDH-s and KAR have the same molecular weight, subunit structure, and tissue distribution. Starch gel electrophoresis followed by histochemical staining using either p-hydroxy-phenylpyruvic acid (HPPA) or malate as the substrate shows that KAR activity comigrates with MDH-s in all species studied except some marine species. Inhibition with malate, the end product of the MDH reaction, substantially reduces or totally eliminates KAR activity. Genetically determined electrophoretic variants of MDH-s seen in the fresh water bony fish of the genus Xiphophorus and the amphibian Rana pipiens exhibited identical variation for KAR, and the two traits cosegregated in the offspring from one R. pipiens heterozygote studied. Both enzymes comigrate with no electrophoretic variation among several inbred strains of mice. Antisera raised against purified chicken MDH-s totally inhibited both MDH-s and KAR activity in chicken liver homogenates. There is no evidence to suggest that any protein besides MDH-s and LDH catalyzes this reaction with the possible exception of the situation in Xiphophorus, in which a third independent zone of HPPA reduction is observed. In most species the activity formerly described as KAR appears to be due to a previously unsuspected activity of MDH-s toward aromatic monocarboxylic alpha-keto acids. In all species examined the KAR activity is associated only with MDH-s; in tissue homogenates the mitochondrial form of MDH (MDH-m) is not detected after electrophoresis using HPPA as a substrate.     

Kinetics of the inhibition of succinate dehydrogenase in bull adrenal cortex by malonate and oxaloacetate

The activity of succinate dehydrogenase from bull adrenal cortex was studied as affected by malonate and oxaloacetate. The both substrate analogs without preincubation (separately and in the mixture) inhibit the enzyme by the competitive type. After a 3 min oxaloacetate preincubation of the enzyme inhibition is of a mixed character. Malonate under these conditions lowers the oxaloacetate effect without changing the type of inhibition. It is supposed that the protective effect is due to a high rate of formation and decay of the enzyme-inhibitory malonate complex.