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.     

Kinetics of the reduction of oxaloacetate catalyzed by mitochondrial malate dehydrogenase of Toxocara canis muscle

1. The reaction of reduction of oxaloacetate to L-malate in the presence of NADH catalyzed by mitochondrial malate dehydrogenase (EC 1.1.1.37) of Toxocara canis muscle has been studied. 2. The data obtained in initial velocity experiments as well as those involving product inhibition suggest that the reaction mechanism is of the sequential type with a kinetically significant ternary complex and in which the coenzymes bind to the free enzyme. 3. The kinetic parameters, including the inhibition constant for NADH were estimated by non-linear regression analysis using the appropriate rate equations.     

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.     

Effects of temperature and humidity on the development of eggs of Toxocara canis under laboratory conditions

The influence of temperature and humidity on the survival and development of Toxocara canis eggs in an in vitro model system was investigated. Two soil samples were inoculated with T. canis eggs and maintained at 3% and 50% humidity and temperatures of 19-24 degrees C. Nine soil samples were inoculated with T. canis eggs of which three samples were kept at 4 degrees C with humidities at 3%, 15%, and 30%; three were maintained at 21 degrees C and three more were incubated at 34 degrees C, and at the same three humidity levels. Samples were monitored every 7 days for a total of 2 months, for the presence and development of eggs. With increasing temperature, the number of eggs undergoing development increased (P<0.01); the number of deformed eggs decreased, the number of infective eggs increased (P<0.01), and egg maturation was accelerated. A decrease in the survival of infective eggs occurred at 34 degrees C. An increase in humidity produced a rise in the number of developed eggs at all three temperatures (P<0.01). This study suggests that elevated temperatures accelerated the development as well as the degradation of eggs of T. canis, whereas the range in humidity was directly correlated with egg development.     

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.     

Binding of mitochondrial malate dehydrogenase to mitoplasts.

The binding of 14C-labelled bovine and porcine malate dehydrogenase (EC 1.1.1.37) to rat liver mitochondria and mitoplasts was examined. The bovine enzyme was found to associate nonspecifically with isolated mitochondria and sonicated mitoplasts. Scatchard plot analysis suggested a specific binding to mitoplasts of the order of 5 pmol malate dehydrogenase per milligram of mitoplast protein. Porcine malate dehydrogenase dimer but not monomer exhibited a similar binding. The results are discussed in relation to the mechanism of uptake of the enzyme by mitochondria after synthesis on cytosolic ribosomes.     

The epidemiology of Toxocara canis

Bred as hunter, companion and pet, the dog has a long and honourable association with man. Yet the domestic dog can host a wide range of parasites - many of which can also infect humans. One of these, the ascarid nematode Toxocara canis (Fig. 1), is of particular interest because of retinal damage that may result from larvae becoming trapped in the eye. At the Hospital for Tropical Diseases in London, about 20-30 patients with toxocariasis are treated annually. Widespread fouling of public parks, playgrounds and pedestrian areas with dog faeces - especially in large cities - is well -recognized as one of the main sources of Toxocara infection. Yet as Stephen Gillespie discusses here, epidemiological indicators vary widely and the risk of infection is often treated too lightly.     

Toxocara canis and the allergic process

The protective effect of infectious agents against allergic reactions has beenthoroughly investigated. Current studies have demonstrated the ability of somehelminths to modulate the immune response of infected hosts. The objective of thepresent study was to investigate the relationship between Toxocaracanis infection and the development of an allergic response in miceimmunised with ovalbumin (OVA). We determined the total and differential blood andbronchoalveolar lavage fluid cells using BALB/c mice as a model. To this end, thelevels of interleukin (IL)-4, IL-5 and IL-10 and anti-OVA-IgE were measured using anELISA. The inflammatory process in the lungs was observed using histology slidesstained with haematoxylin and eosin. The results showed an increase in the totalnumber of leukocytes and eosinophils in the blood of infected and immunised animalsat 18 days after infection. We observed a slight lymphocytic inflammatory infiltratein the portal space in all infected mice. Anti-OVA-IgE levels were detected insmaller proportions in the plasma of immunised and infected mice compared with micethat were only infected. Therefore, we concluded that T. canispotentiates inflammation in the lungs in response to OVA, although anti-OVA-IgElevels suggest a potential reduction of the inflammatory process through thismechanism.     

NADH binding to porcine mitochondrial malate dehydrogenase.

The binding of NADH to porcine mitochondrial malate dehydrogenase in phosphate buffer at pH 7.5 has been studied by equilibrium and kinetic methods. Hyperbolic binding was obtained by fluorimetric titration of enzyme with NADH, in the presence or absence of hydroxymalonate. Identical results were obtained for titrations of NADH with enzyme in the presence or absence of hydroxymalonate, measured either by fluorescence emission intensity or by the product of intensity and anisotropy. The equilibrium constant for NADH dissociation was 3.8 +/- 0.2 micrometers, over a 23-fold range of enzyme concentration, and the value in the presence of saturating hydroxymalonate was 0.33 +/- 0.02 micrometer over a 10-fold range of enzyme concentration. The rate constant for NADH binding to the enzyme in the presence of hydroxymalonate was 3.6 X 10(7) M-1 s-1, while the value for dissociation from the ternary complex was 30 +/- 1 s-1. No limiting binding rate was obtained at pseudo-first order rate constants as high as 200 s-1, and the rate curve for dissociation was a single exponential for at least 98% of the amplitude. In addition to demonstrating that the binding sites are independent and indistinguishable, the absence of effects of enzyme concentration on the KD value indicates that NADH binds with equal affinity to monomeric and dimeric enzyme forms.     

Light and acetate regulate a mitochondrial malate dehydrogenase

A malate dehydrogenase was purified from the unicellular green alga Chlorogonium elongatum Dangeard. The enzyme was localized in the mitochondria by immunogold electron microscopy and was found to be present on the cristae. The concentration of the enzyme is regulated by acetate and light. In cells cultured heterotrophically with acetate as carbon source the activity and the concentration of the enzyme is 5- to 6-fold higher than in autotrophic cells. In mixotrophically cultured cells (light and acetate) the enzyme level attains only half of the value of that in heterotrophic cells. Acetate induces an increase of the enzyme concentration while light has an inhibitory effect on this process.