Schistosoma mansoni: Purification and characterization of malate dehydrogenases

Isoelectric focusing of a homogenate of Schistosoma mansoni, followed by malate dehydrogenase-specific staining, showed the presence of two major and five minor malate dehydrogenase isoenzymes (EC 1.1.1.37), with isoelectric points ranging from 7.3 to 9.5. The malate dehydrogenase isoenzymes were purified by gel filtration, followed by ion-exchange chromatography on DEAE- and CM-cellulose. The isoenzymes could be differentiated by their susceptibility to substrate inhibition. No differences in the Michaelis-Menten constants for substrate were found. One of the isoenzymes is inhibited by 5′-AMP. Further purification of this particular isoenzyme was achieved by affinity chromatography on 5′-AMP-Sepharose 4B. Analysis after subcellular fractionation indicated a mitochondrial origin for this isoenzyme. The mitochondrial isoenzyme (at a recovery of 80%) was purified 218-fold compared to the crude soluble extract, and contained about 40% of the total malate dehydrogenase activity. The enzyme has a molecular weight of 65,500 and showed absolute specificity for l-malic acid, NAD, and NADH. The final preparation has a specific activity of 451 U/mg protein. Physicochemical studies, including binding constants, substrate inhibition, thermostability, and pH optima, demonstrated differences between the mitochondrial and cytoplasmic enzymes. A role for malate dehydrogenase in Schistosoma mansoni metabolism is discussed.     

Mitochondrial malate dehydrogenase from corn : purification of multiple forms

A method to fractionate corn (Zea mays L. B73) mitochondria into soluble proteins, high molecular weight soluble proteins, and membrane proteins was developed. These fractions were analyzed by both sodium dodecyl sulfate-polyacrylamide gel electrophoresis and assays of mitochondrial enzyme activities. The Krebs cycle enzymes were enriched in the soluble fraction. Malate dehydrogenase has been purified from the soluble fraction by a two-step fast protein liquid chromatography method. Six different malate dehydrogenase peaks were obtained from the Mono Q column. These peaks were individually purified using a Phenyl Superose column. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified peaks showed that three of the isoenzymes consisted of different homodimers (I, III, VI) and three were different heterodimers (II, IV, V). Apparent molecular masses of the three different monomer subunits were 37, 38, and 39 kilodaltons. Nondenaturing gel analysis of the malate dehydrogenase peaks showed that each Mono Q peak contained a band of malate dehydrogenase activity with different mobility. These observations are consistent with three nuclear genes encoding corn mitochondrial malate dehydrogenase. Polyclonal antibodies raised against purified malate dehydrogenase were used to identify the gene products using Western blots of two-dimensional gels.     

A 1H nuclear-magnetic-resonance study of the conformation and the molecular dynamics of the glycoprotein cow-colostrum trypsin inhibitor

1. One mitochondrial and one cytoplasmic malate dehydrogenase isoenzyme could be purified from acetate grown cells of the yeast Saccharomyces cerevisiae. 2. The purification procedure uses chromatography on dextran blue columns as an essential step for enrichment, and reverse ammonium sulfate chromatography on celite for isoenzyme separation. 3. The homogeneity of the preparations was established by gel electrophoreses in the presence of sodium dodecylsulfate and by a sedimentation run in the analytical ultracentrifuge. 4. Both enzymes are dimers with a molecular weight of 75 000 for the cytoplasmic and of 68 000 for the mitochondrial enzyme. 5. Amino acid analysis and peptide mapping showed that both enzymes are closely related, but genetically different (true isoenzymes). 6. The cytoplasmic enzyme shows electrophoretic splitting. This is most likely due to post-translational deamination in vivo. 7. Antibodies to both isoenzymes could be obtained in rabbits. The antisera to cytoplasmic malate dehydrogenase were specific for this enzyme. Antisera to mitochondrial malate dehydrogenase react with both isoenzymes. Neither type of antisera precipitated an inactive protein after the glucose-dependent inactivation of cytoplasmic malate dehydrogenase in vivo.     

Determination of NAD Malic Enzyme in Leaves of C(4) Plants : EFFECTS OF MALATE DEHYDROGENASE AND OTHER FACTORS

Malate dehydrogenase may interfere with the assay of NAD malic enzyme, as NADH is formed during the conversion of malate to oxaloacetate. During the present study, two additional effects of malate dehydrogenase were investigated; they are evident only if the malate dehydrogenase reaction is allowed to reach equilibrium prior to initiating the malic enzyme reaction. One of these (Outlaw, Manchester 1980 Plant Physiol 65: 1136-1138) might cause an underestimation of NAD reduction by malic enzyme due to the oxidation of NADH during reversal of the malate dehydrogenase reaction. A second effect may result in overestimation of malic enzyme activity, as Mn²⁺-catalyzed oxaloacetate decarboxylation causes continuing net NADH formation via malate dehydrogenase. These effects were studied by assaying the activity of a partially purified preparation of Amaranthus retroflexus NAD malic enzyme in the presence or absence of purified NAD malate dehydrogenase.     

Interaction between citrate synthase and mitochondrial malate dehydrogenase in the presence of polyethylene glycol

Pig heart citrate synthase and mitochondrial malate dehydrogenase interact in polyethylene glycol solutions as indicated by increased solution turbidity. A large percentage of both enzymes sediments when mixtures of the two in polyethylene glycol are centrifuged, whereas little if any of either enzyme sediments in the absence of the other. The observed interaction is highly specific in that neither cytosolic malate dehydrogenase nor nine other proteins showed evidence of specific interaction with either pig heart citrate synthase or mitochondrial malate dehydrogenase. Escherichia coli citrate synthase did not interact with pig heart citrate synthase, but did show evidence of interaction with pig heart mitochondrial malate dehydrogenase. The relation between enzyme behavior in polyethylene glycol solution and in the mitochondrion and the significance of possible in vivo interactions between citrate synthase and mitochondrial malate dehydrogenase are discussed.     

The malate dehydrogenase isoforms from Trypanosoma brucei: subcellular localization and differential expression in bloodstream and procyclic forms

Trypanosoma brucei procyclic forms possess three different malate dehydrogenase isozymes that could be separated by hydrophobic interaction chromatography and were recognized as the mitochondrial, glycosomal and cytosolic malate dehydrogenase isozymes. The latter is the only malate dehydrogenase expressed in the bloodstream forms, thus confirming that the expression of malate dehydrogenase isozymes is regulated during the T. brucei life cycle. To achieve further biochemical characterization, the genes encoding mitochondrial and glycosomal malate dehydrogenase were cloned on the basis of previously reported nucleotide sequences and the recombinant enzymes were functionally expressed in Escherichia coli cultures. Mitochondrial malate dehydrogenase showed to be more active than glycosomal malate dehydrogenase in the reduction of oxaloacetate; nearly 80% of the total activity in procyclic crude extracts corresponds to the former isozyme which also catalyzes, although less efficiently, the reduction of p-hydroxyphenyl-pyruvate. The rabbit antisera raised against each of the recombinant isozymes showed that the three malate dehydrogenases do not cross-react immunologically. Immunofluorescence experiments using these antisera confirmed the glycosomal and mitochondrial localization of glycosomal and mitochondrial malate dehydrogenase, as well as a cytosolic localization for the third malate dehydrogenase isozyme. These results clearly distinguish Trypanosoma brucei from Trypanosoma cruzi, since in the latter parasite a cytosolic malate dehydrogenase is not present and mitochondrial malate dehydrogenase specifically reduces oxaloacetate.     

Plasma-membrane-bound malate dehydrogenase activity in maize roots

Summary Plasma membranes were isolated and purified from 14-day-old maize roots (Zea mays L.) by two-phase partitioning at a 6.5% polymer concentration, and compared to isolated mitochondria, microsomes, and soluble fraction. Marker enzyme analysis demonstrated that the plasma membranes were devoid of cytoplasmic, mitochondrial, tonoplast, and endoplasmic-reticulum contaminations. Isolated plasma membranes exhibited malate dehydrogenase activity, catalyzing NADH-dependent reduction of oxaloacetate as well as NAD⁺-dependent malate oxidation. Malate dehydrogenase activity was resistant to osmotic shock, freeze-thaw treatment, and salt washing and stimulated by solubilization with Triton X-100, indicating that the enzyme is tightly bound to the plasma membrane. Malate dehydrogenase activity was highly specific to NAD⁺ and NADH. The enzyme exhibited a high degree of latency in both right-side-out (80%) and inside-out (70%) vesicle preparations. Kinetic and regulatory properties with ATP and P_i, as well as pH dependence of plasma-membrane-bound malate dehydrogenase were different from mitochondrial and soluble malate dehydrogenases. Starch gel electrophoresis revealed a characteristic isozyme form present in the plasma membrane isolate, but not present in the soluble, mitochondrial, and microsomal fractions. The results presented show that purified plasma membranes isolated from maize roots contain a tightly associated malate dehydrogenase, having properties different from mitochondrial and soluble malate dehydrogenases.Abbreviations FCR ferricyanide reductase - MDH malate dehydrogenase     

Purification and properties of microbody malate dehydrogenase from Spinacia oleracea leaf tissue

The microbody isoenzyme of malate dehydrogenase (EC 1.1.1.37) from leaves of Spinacia oleracea was purified to a specific activity of 3000 units/mg protein and examined for a number of physical, kinetic, and immunological properties. The purified enzyme has a molecular weight of approximately 70,000 and an isoelectric point of 5.65. Thermal inactivation first order rate constants were 0.068 (35 °C), 0.354 (45 °C), and 2.11 (55 °C) for irreversible denaturation. Apparent millimolar Michaelis constants are 0.34 (NAD, pH 8.5) 0.16 (NADH, pH 7.5), 3.33 (malate, pH 8.5), 0.07 (OAA, pH 6.0), 0.06 (OAA, pH 7.5), and 0.50 (OAA, pH 9.0). The enzyme is stablized by 20% glycerol and can be stored for several months at 4 °C without detectable loss of activity. The purified enzyme is sensitive to the ionic strength of the assay medium exhibiting a pH optimum of 5.65 at high ionic strength and 7.00 at low ionic strength. Rabbit antiserum prepared against the purified microbody MDH shows a single precipitin band on immunodiffusion analysis. Immunological studies indicate that rabbit antiserum prepared against the purified microbody enzyme cross reacts approximately 10% with the mitochondrial isoenzyme of MDH. No cross reaction was shown with the soluble isoenzyme. In general, the data presented in this report tend to support the notion of organelle specific isoenzymes of malate dehydrogenase in higher plant tissues and uniqueness of the microbody form of malate dehydrogenase in particular.     

Malate Oxidation and Cyanide-Insensitive Respiration in Avocado Mitochondria during the Climacteric Cycle

After preparation on self-generated Percoll gradients, avocado (Persea americana Mill, var. Fuerte and Hass) mitochondria retain a high proportion of cyanide-insensitive respiration, especially with α-ketoglutarate and malate as substrates. Whereas α-ketoglutarate oxidation remains unchanged, the rate of malate oxidation increases as ripening advances through the climacteric. An enhancement of mitochondrial malic enzyme activity, measured by the accumulation of pyruvate, closely parallels the increase of malate oxidation. The capacity for cyanide-insensitive respiration is also considerably enhanced while respiratory control decreases (from 3.3 to 1.7), leading to high state 4 rates.

Both malate dehydrogenase and malic enzyme are functional in state 3, but malic enzyme appears to predominate before the addition of ADP and after its depletion. In the presence of cyanide, a membrane potential is generated when the alterntive pathway is operating. Cyanide-insensitive malate oxidation can be either coupled to the first phosphorylation site, sensitive to rotenone, or by-pass this site. In the absence of phosphate acceptor, malate oxidation is mainly carried out via malic enzyme and the alternative pathway. Experimental modification of the external mitochondrial environment in vitro (pH, NAD⁺, glutamade) results in changes in malate dehydrogenase and malic enzyme activities, which also modify cyanide resistance. It appears that a functional connection exists between malic enzyme and the alternative pathway via a rotenone-insensitive NADH dehydrogenase and that this pathway is responsible, in part, for nonphosphorylating respiratory activity during the climacteric.

     

Mitochondrial malate dehydrogenase of watermelon cotyledons: Time course and mode of enzyme activity changes during germination

Summary Specific antibodies were prepared against the purified mitochondrial malate dehydrogenase (EC 1.1.1.37) from cotyledons of watermelon seedlings (Citrullus vulgaris Schrad.). The isoenzyme was assayed by means of quantitative radial immunodiffusion. Cotyledons of ungerminated seeds were found to contain mitochondrial MDH. During the first 4 days of germination the enzyme activity increased threefold finally contributing 16% to the total MDH activity extracted from cotyledon tissue. Isopycnic CsCl density centrifugation was used to investigate the mode of activity increase. After a four-day period of labelling with deuterium oxide and purification of the mitochondrial isoenzyme, a density shift of 0.021kgx1^-1, accompanied by considerable band broadening of the enzyme profile was observed. These findings are evidence for the de novo synthesis of mitochondrial MDH and its relatively slow turnover in germinating seeds.Abbreviations mMDH mitochondrial malate dehydrogenase - D₂O deuterium oxide     

Isolation and characterization of yeast protease B.

Protease B was purified from baker's yeast. The final preparation appeared homogeneous by ultracentrifugation and electrophoresis. The S₂₀, ω value of the enzyme was 3.1 S and its molecular weight was calculated to be 31,000 from the results of sedimentation equilibrium analysis. The amino acid composition of the enzyme was also investigated. The enzyme inactivates phosphogluconate dehydrogenase and uricase, but not malate dehydrogenase, alcohol dehydrogenase, glucose-6-phosphate dehydrogenase or hexokinase.     

Characterization of the Tumorlike (T) Antigen Induced by Type 12 Adenovirus : I. Purification of the Antigen from Infected KB Cells and a Hamster Tumor Cell Line

The T antigen induced by type 12 adenovirus was purified from KB cells infected in the presence of 10(-6)m 5-fluoro-2-deoxyuridine to inhibit synthesis of viral capsid antigens. The antigen was purified approximately 200-fold, and the purified product contained only negligible amounts of host-cell contaminants, as judged by the residual radioactivity from (14)C-labeled uninfected cells which had been added to infected cells at the initiation of the purification. Immunoelectrophoresis indicated that the purified T-antigen preparation contained a single antigenic species. The T antigen from a hamster cell line (HT-1) derived from a type 12 adenovirus-induced tumor was purified by the same procedure. The T antigens from the two different sources were shown to be immunologically similar by use of a rabbit antiserum prepared against the purified T antigen from infected KB cells and sera from hamsters bearing tumors induced by type 12 adenovirus.     

Partial Purification and Characterization of Complex I, NADH:Ubiquinone Reductase, from the Inner Membrane of Beetroot Mitochondria

A NADH dehydrogenase was isolated from an inner membrane-enriched fraction of beetroot mitochondria (Beta vulgaris L.) by solubilization with sodium deoxycholate and purified using gel filtration and affinity chromatography. The NADH dehydrogenase preparation contained a minor ATPase contamination. Beetroot mitochondria were chosen as the isolation material for purifying the enzymes responsible for oxidizing matrix NADH due to the absence of the externally facing NADH dehydrogenase in the variety we have used. The purified NADH dehydrogenase complex catalyzed the reduction of various electron acceptors with NADH as the electron donor, was not sensitive to rotenone inhibition, and had a slow NADPH-ubiquinone 5 reductase activity. The isolated complex contained 14 major polypeptides. It was concluded that the dehydrogenase represented a form of the plant mitochondrial complex I and not the internally facing rotenone-insensitive NADH dehydrogenase found in plant mitochondria because of its complex structure, its cross-reactivity with antisera raised against bovine heart mitochondrial complex I, and the similarity of its kinetics and inhibitor responses to rotenone-sensitive NADH oxidation by beetroot submitochondrial particles.     

Behaviour of some mitochondrial enzymes in ripening tomato fruit

The activities of four mitochondrial enzymes were studied in four stages of ripening tomato fruit. The highest enzyme activity was recorded for malate dehydrogenase followed by cytochrome c oxidase. Succinate dehydrogenase and NADH oxidase levels were low and could only be determined in the green stage of the fruit. However, peaks of various enzyme activities coincided in identical mitochondrial fractions on the sucrose density gradient. Moreover, the levels of malate dehydrogenase and cytochrome c oxidase were constant during the ripening process while the other two enzymes, succinate dehydrogenase and NADH oxidase, declined. This might indicate that mitochondria retain some of their essential functions through the ripening process.     

Rat liver mitochondria contain two immunologically distinct dihydrolipoamide dehydrogenases

We have raised antisera against dihydrolipoamide dehydrogenase. One antigen was isolated from purified bovine kidney pyruvate dehydrogenase complex (PDC). The other antigen was a commercial preparation of porcine heart dihydrolipoamide dehydrogenase (E3) which did not first involve purification of the alpha-keto acid dehydrogenase complex(es). Both antibody preparations cross-reacted with the E3 components of PDC, alpha-ketoglutarate dehydrogenase complex, and branched-chain keto acid dehydrogenase complex. This demonstrates the immunological identity of the E3 components. These sera totally precipitated E3 activity from the purified complexes, from purified preparations of E3, and from extracts of rat heart and kidney mitochondria. The two sera vary in their reaction with rat liver mitochondrial extracts: the anti PDC-E3 serum left residual E3 activity (approximately 50% of the original) that was precipitable by the anti-E3 anti-serum. This indicates that liver contains two immunologically distinct forms of E3. Metabolic assays measuring the differential effects of the two sera on the glycine decarboxylation reaction suggest that the form which is immunologically nonreactive with the anti-PDC-E3 serum could represent the E3 involved in the glycine cleavage system.     

A Novel Malate Dehydrogenase from Ceratonia siliqua L. Seeds with Potential Biotechnological Applications

A novel malate dehydrogenase (MDH; EC 3.1.1.1.37), hereafter MDHCs, from Ceratonia siliqua seeds, commonly known as Carob tree, was purified by using ammonium sulphate precipitation, ion exchange chromatography on SteamLine SP and gel-filtration. The molecular mass of the native protein, obtained by analytical gel-filtration, was about 65?kDa, whereas, by using SDS-PAGE analysis, with and without reducing agent, was 34?kDa. The specific activity of purified MDHCs (0.25?mg/100?g seeds) was estimated to be 188 U/mg. The optimum activity of the enzyme is at pH 8.5, showing a decrease in the presence of Ca²⁺, Mg²⁺ and NaCl. The N-terminal sequence of the first 20 amino acids of MDHCs revealed 95?% identity with malate dehydrogenase from Medicago sativa L. Finally, the enzymatic activity of MDHCs was preserved even after absorption onto a PVDF membrane. To our knowledge, this is the first contribution to the characterization of an enzyme from Carob tree sources.     

Isolation and expression of the gene encoding yeast mitochondrial malate dehydrogenase.

The mitochondrial tricarboxylic acid cycle enzyme malate dehydrogenase was purified from Saccharomyces cerevisiae, and an antibody to the purified enzyme was obtained in rabbits. Immunoscreening of a yeast genomic DNA library cloned into a lambda gt11 expression vector with anti-malate dehydrogenase immunoglobulin G resulted in identification of a lambda recombinant encoding an immunoreactive beta-galactosidase fusion protein. The yeast DNA portion of the coding region for the fusion protein translates into an amino acid sequence which is very similar to carboxy-terminal sequences of malate dehydrogenases from other organisms. In s. cerevisiae transformed with a multicopy plasmid carrying the complete malate dehydrogenase gene, the specific activity and immunoreactivity of the mitochondrial isozyme are increased by eightfold. Expression of both the chromosomal and plasmid-borne genes is repressed by growth on glucose. Disruption of the chromosomal malate dehydrogenase gene in haploid S. cerevisiae produces mutants unable to grow on acetate and impaired in growth on glycerol plus lactate as carbon sources.     

Cell wall-associated malate dehydrogenase activity from maize roots

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1 M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn²⁺ and Cu²⁺ in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn²⁺ and Cu²⁺ in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.     

Organelle-bound malate dehydrogenase isoenzymes are synthesized as higher molecular weight precursors

Biosynthesis of malate dehydrogenase isoenzymes was studied in cotyledons of watermelons (Citrullus vulgaris Schrad., var. Stone Mountain). The glyoxysomal and mitochondrial isoenzymes are synthesized as higher molecular weight precursors which can be immunoprecipitated by mono-specific antibodies from the products of in vitro translation in reticulocyte lysates programed with cotyledonary mRNA and with the same size from enzyme extracts of pulse-labeled cotyledons. During translocation from the cytosol into the organelles, processing takes place. An 8 kilodalton extra sequence is cleaved from the glyoxysomal precursor and a 3.3 kilodalton extra sequence from the mitochondrial precursor producing the native subunits of 33 and 38 kilodaltons, respectively. The data support a post-translational translocation of the organelle-destined malate dehydrogenase isoenzymes. The in vitro translation of the cytosolic malate dehydrogenase I yields a product which has the same molecular weight as the subunit of the native isoenzyme (39.5 kilodaltons).     

RESPIRATION AND PROTEIN SYNTHESIS IN ESCHERICHIA COLI MEMBRANE-ENVELOPE FRAGMENTS : I. Oxidative Activities with Soluble Substrates

This paper describes experiments conducted with membranous and soluble fractions obtained from Escherichia coli that had been grown on succinate, malate, or enriched glucose media. Oxidase and dehydrogenase activities were studied with the following substrates: nicotinamide adenine dinucleotide, reduced form (NADH), nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), succinate, malate, isocitrate, glutamate, pyruvate, and α-ketoglutarate. Respiration was virtually insensitive to poisons that are commonly used to inhibit mitochondrial systems, namely, rotenone, antimycin, and azide. Succinate dehydrogenase and NADH, NADPH, and succinate oxidases were primarily membrane-bound whereas malate, isocitrate, and NADH dehydrogenases were predominantly soluble. It was observed that E. coli malate dehydrogenase could be assayed with the dye 2,6-dichlorophenol indophenol, but that porcine malate dehydrogenase activity could not be assayed, even in the presence of E. coli extracts. The characteristics of E. coli NADH dehydrogenase were shown to be markedly different from those of a mammalian enzyme. The enzyme activities for oxidation of Krebs cycle intermediates (malate, succinate, isocitrate) did not appear to be under coordinate genetic control.