Soluble Malate Dehydrogenase Research Articles

Verapamil caused biochemical alteration, DNA damage, and expression of hepatic stress-related gene biomarkers in Nile tilapia, Oreochromis niloticus

Impacts of long-term exposure to sub-lethal verapamil (VRP) at 0.14, 0.28, and 0.57 mg L−1 were examined for 60 days to find the effects on activities of metabolic catalysts as well as molecular reactions in Nile tilapia. Alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase activities in the liver increased significantly (p < 0.05) at high concentration of the drug while there was no significant difference in the activities of soluble phosphatases, corrosive phosphatase, and malate dehydrogenase in comparison with the control. Enzymes of gluconeogenesis, including fructose 1,6-bisphosphatase and glucose-6-phosphatase, were induced while RNA/DNA proportion in fish liver decreased significantly in the fish exposed to the VRP from the 15th day onwards. It caused DNA damage and upregulated expression of hsp70, cat, and sod in the liver, which were found to be correlated with concentration of the drug. Prolonged exposure to sub-lethal VRP could change activities of metabolic catalysts, causing harm to DNA and upregulating stress-related genes in the fish which suggested use of these biomarkers as early cautioning signs about the existence of the drug in aquatic ecosystems.

Localization of the genetic unit controlling the photoperiodic adult diapause in Drosophila littoralis

The linkage of the locus Critical daylength for photoperiodic diapause (Cdl) was studied in relation to six other autosomal loci in Drosophila littoralis. The markers distinguishing the two stocks used were Cdl12 from Caucasus and Cdl19 from Oulu. At 16°C and light-dark cycle of 15:9, Cdl12/19 heterozygotes are in diapause and the Caucasus allele thus behaves as a recessive. The other markers were curled wings (cu), and electrophoretically detectable alleles of loci coding for malic enzyme (Me), soluble malate dehydrogenase (Mdh), phosphoglucomutase (Pgm), acid phosphatase (Acph-1), and α-esterase (Est-4). Two linkage groups emerged. The first group contains the loci Me -6- cu -7- Est-4 (the figures refer to crossing-over percentages), and it represents the second chromosome. The second linkage group is Pgm -14- Mdh -27- Cdl, which is deduced to be in the large metacentric autosome. This contains the third and fourth chromosomes of the basic chromosomal set of the D. virilis group. Acph-1 is not linked with any of the other markers studied.

Limited Mitochondrial Permeabilization Is an Early Manifestation of Palmitate-induced Lipotoxicity in Pancreatic β-Cells

Involvement of the mitochondrial permeability transition (MPT) pore in early stages of lipotoxic stress in the pancreatic beta-cell lines MIN6 and INS-1 was the focus of this study. Both long term (indirect) and acute (direct) effects of fatty acid (FA) application on beta-cell susceptibility to Ca(2+)-induced MPT induction were examined using both permeabilized and intact beta-cells. Long term exposure to moderate (i.e. below cytotoxic) levels of the saturated FA palmitate sensitized beta-cell mitochondria to MPT induced by Ca(2+). Long term exposure to palmitate was significantly a more efficient inducer of MPT than the unsaturated FA oleate, although upon acute application both caused similar MPT activation. Application of antioxidants, inhibitors of the ceramide pathway, or modifiers of membrane fluidity did not protect beta-cell mitochondria from FA exposure. However, significant protection was provided by co-application of the unsaturated FA oleate in a phosphatidylinositol 3-kinase-dependent manner. Characterization of MPT pore opening in response to moderate palmitate treatment revealed the opening of a unique form of MPT in beta-cells as it encompassed features of both low and high conductance MPT states. Specifically, this MPT showed solute selectivity, characteristic of a low conductance MPT; however, it affected mitochondrial respiration and membrane potential in a way typical of a high conductance MPT. Activation of the full-size/high conductance form of MPT required application of high levels of FA that reduced growth and initiated apoptosis. These findings suggest that in the beta-cell, MPTs can act as both initiators of cell death and as versatile modulators of cell metabolism, depending on the mode of the MPT pore induced.

Soluble malate dehydrogenase of Geophagus brasiliensis (Cichlidae, Perciformes): isolated isoforms and kinetics properties

Kinetic properties and thermal stabilities of Geophagus brasiliensis skeletal muscle unfractionated malate dehydrogenase (MDH, EC 1.1.1.37) and its isolated isoforms were analyzed to examine a possible sMDH-B* locus duplication in a fixation process influenced by genetic drift. Two optimal pHs were detected: 7.5 for AB1 unfractionated muscle phenotype and its B1 isoform, and 8.0 for AB1B2 unfractionated muscle phenotype, A and B2 isoforms. While G. brasiliensis A isoform could be characterized as thermostable, the duplicated B isoform cannot be assumed as thermolabile. Km values for isolated B2 isoforms were 1.6 times lower than for B1. A duplication event in progress best explains the electrophoretic six-band pattern detected in G. brasiliensis, which would be caused by genetic drift.

Thermal stability of soluble malate dehydrogenase isozymes of subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes

Electrophoretic thermostability tests of soluble malate dehydrogenases (sMDH) isozymes in tissue extracts of 21 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes showed three distinct results. The first, characterized by thermal stability of the slowest-migrating band or A-isoform, was detected in 52% of all species. The second, exhibited in 29% of the species analyzed, had a bidirectionally divergent pattern of their sMDH locus expression, and was characterized by a nondivergent thermostability pattern of both sMDH-A* and B*. In the third category, obtained in 19% of the species studied (the four Siluriformes species), thermostability of the fastest-migrating bands, or B-isoforms, was observed. Comparison of the effects of habitat temperature on the activity of paralogous and orthologous isoforms in tissue extracts of two of these species with different thermostability properties (Leporinus friderici - thermostable sMDH-A*, and Pimelodus maculatus - reverse thermostability properties or reverse electrophoretic pattern), collected during winter and summer months, showed that A and B subunits were present at different quantitative levels and their activities were nearly season independent. Differences in susceptibility to temperature (50°C) of both sMDH loci from tissue extracts of these species were found. In P. maculatus, these susceptibilities helped strengthen one of the hypotheses: the reverse thermostability pattern, where the fastest-migrating band or the B-isoform was the thermostable sMDH. Thus, temperature differences among orthologous homologues of sMDH seem to have occurred in these acclimatized species, where the fastest-migrating band, usually muscle specific and thermolabile in most teleosts, appeared in P. maculatus as the thermostable isoform.

Plasmalemma-associated malate dehydrogenase activity in onion root cells

Plasma membrane vesicles isolated from onion roots showed oxaloacetate reductase activity as well as other oxidoreductase activities. Purification and further sequencing showed that the protein responsible for the activity is a 40 kDa protein which corresponds to the cytosolic soluble malate dehydrogenase. However, the activity remained bound to the membrane after repeated freezing and thawing cycles and further washing, excluding a cytosolic contamination as the source of the activity. Furthermore, a second 28 kDa protein has been copurified together with the 40 kDa protein. The plasmalemma oxaloacetate reductase activity shows both donor and acceptor sites located towards the cytoplasmic side of the plasma membrane. This enzyme catalyzed the oxidation of NADH by oxaloacetate and the reduction of NAD+ by malate in the presence of an oxaloacetate-withdrawing system. We conclude that a significant amount of the cytosolic malate dehydrogenase can be specifically attached to the cytosolic face of the plasmalemma. A possible role in a putative malate shuttle associated to the plasma membrane is discussed.

Malate dehydrogenase in subtropical fish belonging to the orders characiformes, siluriformes and perciformes I. Duplicate gene expression and polymorphism

1. 1. Electrophoretic analysis of the soluble malate dehydrogenase (sMDH) from 22 subtropical fish belonging to the orders Characiformes, Siluriformes and Perciformes, collected in 10 reservoirs of São Paulo State and in two lakes of Minas Gerais State, Brazil, indicates that at least two sMDH loci, MDH-A ∗ and MDH-B ∗, are active. In addition to this latter locus, in Hoplias malabaricus (Erythrinidae, Characiformes), a MDH-A 1,3 ∗ isoloci is proposed in order to explain the six-banded pattern detected in all the individuals screened. 2. 2. In attempting to explain the multiplicity of compounds detected in 87% of the Geophagus brasiliensis (Cichlidae, Perciformes) specimens analyzed, three hypotheses are proposed: the event of duplication in processing the presence of three loci with a null allele within the MDH-B ∗, and overdominance. 3. 3. In 87% of the species here studied, a bidirectionally divergent pattern of expression of the sMDH loci was observed, in which the least anodal isozyme A 2 predominated in liver, and the most anodal isozyme B 2 predominated in skeletal muscle. In two siluriform species, Pimelodela gracilis and Hypostomus regani, and in one perciform, Tilapia rendalli, a unidirectionally divergent pattern, in which the isozyme A 2 predominated in every tissue analyzed, was observed. 4. 4. Polymorphism in at least one of the sMDH loci was detected in 9% of the species studied here: Leporinus friderici (Characiformes) at the MDH-A ∗ and P. gracilis at both sMDH loci. In L. friderici and Pimelodus maculatus (Siluriformes), rare alleles at the MDH-B ∗ locus were detected. Polymorphism at the mitochondrial locus was detected in Tilapia rendalli.

Membrane-bound malate dehydrogenase in mitochondria from the alga Cyanidium caldarium

Although in some organisms part of the mitochondrial malate dehydrogenase (EC 1.1.1.37) is membrane-bound, this enzyme is generally regarded as a matrix protein. The present paper describes a malate dehydrogenase from the thermo- and acidophilic alga Cyanidium caldarium, that is tightly bound to the mitochondrial membrane. Data obtained from organelle separations on sucrose density gradients, as well as polyacrylamide gel electrophoresis of crude and purified enzyme preparations, indicate that no malate dehydrogenase is present in the mitochondrial matrix. Part of the malate dehydrogenase on the mitochondrial membranes could be solubilized by treatment with deoxycholate. The solubilized enzyme showed a thermostability and electrophoretic mobility different from that of the soluble (cytosolic) malate dehydrogenase.

Isozymes of soluble malate dehydrogenase (s-MDH) in fishes of the subclasses chondrichthyes and actinopterygii

1. 1. A recopilation of species of fishes used for the study of s-MDH isozymes was made. 2. 2. s-MDH of most fishes was shown to be codified by two gene loci. However, there are fishes with a more complex pattern, with s-MDH composed of subunits codified in three or four gene loci. 3. 3. s-MDH isozymes from ectothermic species proved useful in the study of biochemical adaptation to the environment.

Cellular distribution, purification and electrophoretic properties of malate dehydrogenase in Trichuris ovis and inhibition by benzimidazoles and pyrimidine derivatives

High levels of malate dehydrogenase were found in Trichuris ovis. Two molecular forms of the enzyme, of different cellular location and electrophoretic pattern, were isolated and purified. The activity of soluble malate dehydrogenase was greater than that of mitochondrial malate dehydrogenase. Both forms also displayed different electrophoretic profiles in comparison with purified extracts from goat ( Caprahircus) liver. Substrate concentration directly affected enzyme activity. Host and parasite malate dehydrogenase activity were both inhibited by a series of benzimidazoles and pyrimidine-derived compounds, some of which markedly reduced parasite enzyme activity, but not host enzyme activity. Percentage inhibition by some pyrimidine derivatives was greater than that produced by benzimidazoles.

Adaptative features of enzymes from family sciaenidae (perciformes)—I. studies on soluble malate dehydrogenase ( s-MDH) and creatine kinase (CK) of fishes from the south coast of uruguay

1. 1. Electrophoretic analysis of the soluble malate dehydrogenase ( s-MDH) and creatine kinase (CK) isozyme patterns of three species of temperate fishes (Sciaenidae, Perciformes) indicates at least two loci for s-MDH, Mdh-A and Mdh-B, and four CK, Ck-A, Ck-B, Ck-C and Ck-D. 2. 2. The subunits encoded by these loci occurred at different levels in different tissues and organs analyzed. 3. 3. Through electrophoretic analysis the products of these loci showed different behaviour to changes in temperature. 4. 4. Relative activities of s-MDH and CK isozymes were compared by Klebe's (1975) method to determine pattern of divergence of duplicated gene expression in the three studied species.

Some genetic markers in Castillian populations.

Haptoglobin, phosphoglucomutase 1 and 6-phosphogluconate dehydrogenase gene frequencies have been found for some Castillian provinces. The NADH diaphorase I, superoxide dismutase, lactate dehydrogenase, soluble oxaloacetate transaminase, soluble malate dehydrogenase, carbonic anhydrase I and carbonic anhydrase II are non-polymorphic red cell enzymes in Caucasoids and we have verified this in Castillian samples.

Equine marker genes: Polymorphism for soluble erythrocyte malic enzyme.

Polymorphism of equine erythrocyte malic enzyme is detactable on starch gel electrophoresis. The frequency of ME1S was 0.06 in 667 Standardbred and 0.09 in 85 Thoroughbred horses. No genetically determined electrophoretic variation in soluble malate dehydrogenase was detected.

The Reduction of Nicotinamide Adenine Dinucleotide by Mitochondria Isolated from Helianthus tuberosus

Intact mitochondria isolated from Jerusalem artichoke tubers oxidize both malate and citrate. Both substrates were equally effective in reducing the endogenous pool of pyridine nucleotides in the presence of respiratory inhibitors. Only malate, and not citrate, was capable of reducing exogenous NAD+ under similar assay conditions. The rate at which malate reduced added NAD+ was biphasic; the initial rapid phase was inhibited by the accumulation of oxaloacetate while the velocity of the second, slower, phase was found to be insensitive to accumulated oxaloacetate. The addition of the detergent Decon 90 to intact mitochondria stimulated both the rapid and slow phases of NAD+ reduction and failed to convert the biphasic rate into a constant rate. Decon 90 was found to cause inhibition of soluble malate dehydrogenase. Extensive efforts to purify the mitochondria using sucrose density gradients failed to remove all of the soluble malate dehydrogenase from the preparation of mitochondria and approximately 3% of the total malate dehyrogenase present in the

Changes in isoenzymes of soluble malate dehydrogenase during germination of mung bean (Phaseolus aureus Roxb.) under salt stress

Seeds of mung bean (Phaseolus aureusRoxb.) cv. Pusa Baisakhi were surface sterilized and sown both in Petri dishes and sand culture containing aqueous solutions of four different saltsviz. NaCl, KC1, Na2SO4 and K2SO4 each at 5 and 10 m ώ-1 cm-1. Malate dehydrogenase (MDH) isoenzymes were studied in different plant parts of mung bean at suitable intervals during germination under four different salts. In cotyledons, 96 h after sowing only one isoenzyme was left in control as compared to three under salt treatment. In the embryo axis, 96 h after sowing, sulphate salts resulted in the disappearance of isoenzymes with R1 0.43 and 0.62, whereas isoenzyme with R1 0.62 was missing only at a higher concentration of chloride salts. Chloride salts also resulted in the disappearance of band with R1 0.15, both in the embryo axis and leaves. However, in the roots the isoenzymic pattern remains the same with all the salt treatments.

Duplicated cytosolic malate dehydrogenase genes in Zea mays

Six inbred lines of Zea mays expressing different soluble (cytosolic) malate dehydrogenase (sMDH) zymogram phenotypes were analyzed genetically. sMDH was found to be coded for by unlinked duplicated loci in four of these inbred lines. The remaining two lines were found not to possess these duplicated loci. Furthermore, the duplicated loci, sMdh1 and sMdh2, have been found to be located on different chromosomes: sMdh1 on chromosome 1L linked to Amp1, and sMdh2 on chromosome 5S linked to Cat1 and Amp3. The importance of finding sMDH encoded by duplicated loci is discussed in relation to the role of chromosomal rearrangements, the relationship between the cytoplasmic and mitochondrial enzymes, and the evolution of Z. mays.

Changes in the properties of fetal rat liver during organ culture.

Explants of fetal rat liver maintained in organ culture lost about 40% of their mass in 42 hr of incubation as a result of decrease in blood cells and hepatocytes. Proteins from the cytosol and particulate elements of the tissue were found in the culture medium. About 60% of this protein was degraded to peptides during culture. The transfer of malate and lactate dehydrogenases from tissue to medium paralleled that of proteins. Glutamate dehydrogenase was lost from the mitochondria and in part leaked through the cell membrane into the medium. Net loss of activity of the three enzymes occurred, probably as a consequence of proteolytic degradation. Of 12 enzymes in liver tissue, the specific activities of eight—soluble malate dehydrogenase, glutamate dehydrogenase, succinate dehydrogenase, phosphopyruvate carboxylase, hexosediphosphatase, glucose-6-phosphatase, tyrosine, aminotransferase, and alanine aminotransferase—were unchanged or increased. Glycogen synthetase, aspartate aminotransferase, pyruvate kinase, and lactate dehydrogenase decreased. Although changes in membrane permeability may have had some influence on the results reported, the predominant effect was due to loss of protein from tissue as a result of discharge of total contents of some of the cells into the medium. The residual explanted tissue retained its structural integrity. It is concluded that fetal rat liver in organ culture provides a suitable model system for controlled studies with this organ in vitro.

Adaptation of Drosophila enzymes to temperature—II. Supernatant and mitochondrial malate dehydrogenase

The observed temperature-dependent K m patterns were similar among species ( Drosophila) from the same habitat and different among species from different habitats. The Q 10 values in general reflected temperature dependent changes in K m. The observed convergence in terms of K m and Q 10 values and assay temperature strongly suggests that natural selection is operating at the malate dehydrogenase locus in the genus Drosophila with the temperature being one of the mediating agents. Our data, based on biochemical analysis, show that mitochondrial malate dehydrogenase seems to be a ‘conservative’ enzyme from the evolutionary point of view. This enzyme isolated either from temperate or tropical species exhibits ‘symptoms’ of tropical enzyme in terms of K m vs. temperature. The fact that mitochondrial malate dehydrogenase and soluble malate dehydrogenase do not react coordinatly, bears on the significance of the existence of two forms of the enzyme.

The Oxidation of Malate by Isolated Turnip (Brassica napusL.) Mitochondria

Oxaloacetic acid (OAA) and fluoro-oxaloacetic acid (FOAA) are both specific inhibitors of malate dehydrogenases. Exogenous OAA penetrates the mitochondria rapidly, being converted to pyruvate, citrate, or malate depending on the reaction conditions, with the relief of the inhibition, whereas inhibition by FOAA is permanent. Exogenous OAA removal has been used as a model for endogenous OAA removal by measuring the time taken for the inhibition to be relieved in the presence of various inhibitors and substrates. Hydroxymalonate behaves primarily as a malic enzyme inhibitor while butylmalonate acts as an inhibitor of malate transport in intact mito chondria, although at high concentrations both compounds inhibit the malate dehydrogenase. The NAD associated with the various enzymes behaves in a complex compartmented manner. It is concluded that the oxidation of malate takes place mainly via the membrane-bound malate dehydrogenase, linked through all three phosphorylation sites. The OAA produced is removed by the combined action of the soluble malate dehydrogenase and malic enzyme of the matrix. Reducing equivalents from the malic enzyme can also gain access to the full respiratory chain and utilize all three phosphorylation sites. Evidence is presented for malic enzyme activity on the outside of the inner membrane and in the matrix compartment.

The effects of prednisolone on the rat enterocyte at a subcellular level.

1. Enterocytes, isolated from the proximal jejunum and distal ileum of normal and prednisolone-treated rats, were homogenized and fractionated by isopycnic centrifugation on sucrose density gradients. The distributions of marker enzymes for the principal subcellular organelles, RNA and protein were determined and related to the activities per enterocyte. 2. In enterocytes fromthe jejunum and ileum of prednisolone-treated animals the activities of particulate brush-border enzymes and of both soluble and mitochondrial malate dehydrogenase were increased compared with those of the control system. The equilibrium density of the brush borders was enhanced in the prednisolone-treated jejunum. The modal densities of the other organelles were unaltered by prednisolone administration. 3. There was a large increase in the total RNA content of enterocytes from the jejunum and ileum of prednisolone-treated animals. This was predominantly associated with a distinct particulate component, indicative of a proliferation of the rough endoplasmic reticulum and consistent with an enhanced rate of protein synthesis. 4. Studies of latent brush-border enzyme activities, the mechanical fragility of isolated brush borders and electron microscopy suggest that steroid administration results in no marked alterations in the gross conformation of the brush-border membrane or in the orientation of the enzymes within the membrane.