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1.
The activity and properties of malate dehydrogenase (MDH; EC 1.1.1.37) and of "malic" enzyme (EC 1.1.1.40) in cytosole of the trematode C. ijimai were determined. The activity of MDH directed to oxaloacetate formation was shown to be 14 times and maximum velocity 13 times lower than that of the reverse reaction. The apparent KM was one order higher in the direct reaction. This confirms the possibility of glycolytic pathway in C. ijimai via CO2 fixation into phosphoenolpyruvate to form oxaloacatate which is readily eliminated by active MDH. The presence of "malic" enzyme in C. ijimai testifies to the occurrence of different pathways of succinate formation in this species.  相似文献   

2.
  • 1.1. In the mitochondria of chicken liver cells there is lactate dehydrogenase activity that catalyses the reduction of the oxaloacetate by the NADH.
  • 2.2. The presence of lactate dehydrogenase in the malate dehydrogenase preparations causes an apparent activation in the double-reciprocal plot at high oxaloacetate concentrations that depends on the lactate dehydrogenase/malate dehydrogenase ratio in the preparation.
  • 3.3. The separation of the two molecular forms of chicken liver mitochondrial malate dehydrogenase, free from lactate dehydrogenase, is described.
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3.
Using site-directed mutagenesis on the lactate dehydrogenase gene from Bacillus stearothermophilus, three amino acid substitutions have been made at sites in the enzyme which we suggest in part determine specificity toward different hydroxyacids (R-CHOH-COOH). To change the preferred substrates from the pyruvate/lactate pair (R = -CH3) to the oxaloacetate/malate pair (R = -CH2-COO-), the volume of the active site was increased (thr 246----gly), an acid was neutralized (asp-197----asn) and a base was introduced (gln-102 - greater than arg). The wild type enzyme has a catalytic specificity for pyruvate over oxaloacetate of 1000 whereas the triple mutant has a specificity for oxaloacetate over pyruvate of 500. Despite the severity and extent of these active site alterations, the malate dehydrogenase so produced retains a reasonably fast catalytic rate constant (20 s-1 for oxaloacetate reduction) and is still allosterically controlled by fructose-1,6-bisphosphate.  相似文献   

4.
Various concentration of fructose-1.6-diphosphate, malate, oxaloacetate, creatine phosphate, ATP, ADP and AMP were studied for their effect on the activity of A4-and B4-isoenzymes of lactate dehydrogenase (LDH, EC 1, 1. 1. 27) produced from skeletal muscles and unfertilized egg cells of Misgurnus fossilis in the reactions of lactate oxidation and pyruvate reduction. It was found that oxaloacetate, creatine phosphate, ADP and AMP decreased the activity of A- and B-type isoenzymes to a different extent. The value of the inhibitory action depended not only on the concentration of the substances and subunit composition of the isoenzymes but also depended on the direction of the reaction they catalyse. Malate and fructose-1.6-diphosphate did not inhibit the activity of A4 isoenzyme in the lactate oxidation and malate and ATP did not influence the activity of the former and of B4-isoenzymes in this reaction. At the same time malate, fructose-1.6-diphosphate and ATP decreased the activity of the investigated isoenzymes in the pyruvate reduction reactions.  相似文献   

5.
The hare heart mitochondrial malate dehydrogenase (mMDH) was established to have a much higher electrophoretic mobility than the corresponding enzyme from the rabbit heart. Differences of kinetic properties of both mMDH and cytoplasmic malate dehydrogenase (cMDH) from these two sources were shown. The hare heart mMDH and cMDH isoenzymes have a higher affinity to malate (in direct reaction) and oxaloacetate and NADH (in reverse reaction), i.e., they have lower K M values in comparison with the isoenzymes from the rabbit heart. Malate dehydrogenase seems to operate more effectively in the hare heart, which might be important in adaptive and evolutionary aspects.  相似文献   

6.
Malate dehydrogenase: a model for structure, evolution, and catalysis.   总被引:11,自引:0,他引:11       下载免费PDF全文
Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.  相似文献   

7.
Maria Luisa Sagrist  Jorge Bozal 《Biochimie》1987,69(11-12):1207-1215
Chicken liver microsomal fractions show lactate and malate dehydrogenase activities which behave differently with respect to successive extractions by sonication in 0.15 M NaCl, 0.2% Triton X-100 and 0.15 M NaCl, respectively. The Triton X-100-treated pellet did not show malate dehydrogenase activity but exhibited a 10-fold increase in lactate dehydrogenase activity with respect to the sonicated pellet. Total extracted lactate and malate dehydrogenase activities were, respectively, 7.5 and 1.7 times higher than that in the initial pellet. Different isoenzyme compositions were observed for cytosoluble and microsomal extracted lactate and malate dehydrogenases. When the ionic strength (0-500 mM) or the pH values (6.1-8.7) of the media were increased, an efficient release of lactate dehydrogenase was found at NaCl 30-70 mM and pH 6.6-7.3. Malate dehydrogenase solubilization under the same conditions was very small, even at NaCl 500 mM, but it attained a maximum in the 7.3-8.7 pH range. Cytosoluble lactate dehydrogenase bound in vitro to 0.15 M NaCl-treated (M2) and sonicated (M3) microsomal fractions but not to the crude microsomal fraction (M1). Particle saturation by lactate dehydrogenase occurred with M2 and M3, which contained binding sites with different affinities. Cytosoluble malate dehydrogenase did not bind to M1, M2 and M3 fractions, however, a little binding was found when purified basic malate dehydrogenase was incubated with M2 or M3 fractions.  相似文献   

8.
The malate dehydrogenase activity (EC 1.1.1.37), present in the cytoplasm of Pisum sativum root nodules, can be separated by ion-exchange chromatography into four different fractions. Malate dehydrogenase activity present in the cytoplasm of roots elutes mainly as a single peak. During nodule development an increase in malate dehydrogenase activity per gram of material was observed. This increase occurred concomitantly with the increase in nitrogenase activity. The kinetic properties of the separated malate dehydrogenases of root nodule cytoplasm and root cytoplasm were studied. The Km values for malate (2.6 mM), NAD+ (27 microM), oxaloacetate (18 microM) and NADH (13 microM) of the dominant form of the root nodule cytoplasm are much lower than those of the dominant malate dehydrogenase root form (64 mM, 4.4 mM, 89 microM and 70 microM respectively). Binding of malate by the enzyme-NADH complex from root nodules results in an abortive complex, thereby blocking the further reduction of oxaloacetate by NADH. The dominant root malate dehydrogenase does not form the abortive complex. From the kinetic data it is concluded, first, that the root nodule forms of the enzyme are capable of catalysing at a high rate the reduction of oxaloacetate, to meet the demands for malate governed by the bacteroid and the infected plant cell. The second conclusion, drawn from the kinetic data, is that under physiological conditions the conversion of oxaloacetate can be controlled just by the malate concentration. Consequently the major root nodule forms of malate dehydrogenase are able to allow a high flux of malate production from oxaloacetate but also to establish a sufficient oxaloacetate concentration necessary for the assimilation and transport of fixed nitrogen.  相似文献   

9.
1. When [2-(14)C]pyruvate is injected into rats the C3-position of liver glutamate becomes more heavily labelled than the C2-position, thus establishing that oxaloacetate and fumarate are not in equilibrium in rat liver mitochondria in vivo. The amount of disequilibrium was shown to be simply related to the value that the C3-label/C2-label ratio would have were no label recycled. This ratio, z, was calculated for post-absorptive rats in environmental temperatures of 20 degrees and 30 degrees C from determinations of the distribution of label within glutamate 1, 3 and 10min after intravenous injection of [2-(14)C]pyruvate. The values of z (best estimate and range) were 1.65 (1.60-1.69) in rats at 20 degrees C and 2.43 (2.23-2.63) in rats at 30 degrees C. These values of z imply the following rates of interconversion in mitochondria of fumarate and oxaloacetate (in terms of the oxaloacetate-->citrate flux, R) in rats at 20 degrees C: [Formula: see text] and in rats at 30 degrees C: [Formula: see text] 2. The kinetic parameters of malate dehydrogenase and fumarate hydratase and the intramitochondrial concentrations of NAD(+) and NADH under (as far as could be judged) conditions in vivo were collated. From them and the best estimates of R now available were calculated the rates of interconversion of fumarate, malate and oxaloacetate required to give the found values of z. These rates showed that the fumarate hydratase reaction was nearly in equilibrium, but that the malate dehydrogenase reaction was considerably out of equilibrium. The calculations also led to the following conclusions. 3. In livers of rats at 20 degrees and 30 degrees C mitochondrial malate concentrations were respectively about 5 and 1.5 times mean cellular concentrations. 4. Mitochondrial oxaloacetate concentrations were less than 0.2 of the mean cellular concentrations. They were also only 0.65 and 0.55 of the equilibrium concentrations for the malate dehydrogenase reaction in rats at 20 degrees and 30 degrees C respectively. 5. Malate dehydrogenase activity was low because of the very low oxaloacetate concentrations in the mitochondria and the very small fraction of the enzyme complexed with NAD(+), i.e. in each direction one substrate concentration was very sub-optimal.  相似文献   

10.
Myocardium and skeletal muscle of white rats have a number of specific features in metabolism of carbohydrates. The skeletal muscle is characterized by high intensity of glycolytic processes and glycolytic substrate phosphorylation, that is testified to by the activity of the terminal glycolysis stage enzymes (pyruvate kinase, lactate dehydrogenase, its isoenzyme spectrum) and by the content of lactate and pyruvate metabolites. In contrast to skeletal muscles, the activity of NAD-dependent malate dehydrogenase in the myocardium is significant both in cytoplasm and in mitochondria. This activity corresponds to a high level of malate and oxaloacetate metabolites and to the activity of NADP-dependent malate dehydrogenase, playing a connective role between glycolysis, the cycle of tricarboxylic acids and glyconeogenesis. Phosphoenolpyruvate carboxykinase, catalyzing the transformation of cytoplasmatic oxaloacetate into phosphoenolpyruvate is more active in the skeletal muscles where the intensity of the tricarboxylic acids cycle reactions is lower and the activity of glycolysis is higher than that of myocardium.  相似文献   

11.
The limiting factors of the involvement of malate dehydrogenase in mitochondrial malate oxidation were investigated by using Percoll-purified potato tuber mitochondria. The respective roles of reduced pyridine nucleotides, oxaloacetate, and adenine nucleotides were studied under conditions of high or low phosphorylation potential (Pi + ADP/ATP ratio). Under conditions of high phosphorylation potential, the limitation of malate dehydrogenase activity was caused by the accumulation of oxaloacetate in the medium. In the absence of ADP (phosphorylation potential close to zero), ATP was responsible for the inhibition of malate dehydrogenase activity rather than oxaloacetate or reduced pyridine nucleotides.  相似文献   

12.
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.  相似文献   

13.
Trypanosoma cruzi, the protozoan parasite causing Chagas disease, contains a novel aromatic alpha-hydroxy acid dehydrogenase. This enzyme is responsible, together with tyrosine aminotransferase, for the catabolism of aromatic amino acids, which leads to the excretion of aromatic lactate derivatives into the culture medium. The gene encoding the aromatic alpha-hydroxy acid dehydrogenase has been cloned through a combined approach using screening of an expression genomic library with antibodies, peptide sequencing and PCR amplification. Its sequence shows high similarity to the cytosolic malate dehydrogenases. However, the enzyme has no malate dehydrogenase activity. The gene seems to be present in a single copy per haploid genome and is differentially expressed throughout the parasite's life cycle, the highest levels being found in the insect forms of T. cruzi. The purified recombinant enzyme, expressed in Escherichia coli, was unable to reduce oxaloacetate and had kinetic constants similar to those of the natural aromatic alpha-hydroxy acid dehydrogenase. Sequence comparisons suggest that the aromatic alpha-hydroxy acid dehydrogenase derives from a cytosolic malate dehydrogenase no longer present in the parasite, made redundant by the presence of a glycosomal malate dehydrogenase as a member of a shuttle device involving the mitochondrial isoenzyme.  相似文献   

14.
The rate of phosphoenolpyruvate carboxylase activity measured through the conventional coupled assay with malate dehydrogenase is underestimated due to the instability of oxaloacetate, which undergoes partial decarboxylation into pyruvate in the presence of metal ions. The addition of lactate dehydrogenase to the conventional assay allows the reduction of pyruvate formed from oxaloacetate to lactate with the simultaneous oxidation of NADH. Then, the enzymic determination of substrate and products shows that the combined activities of malate dehydrogenase and lactate dehydrogenase account for all the phosphoenolpyruvate consumed. The net result of the improved assay is a higher Vmax with no apparent effect on Km. The free divalent cation concentration appears to be the major factor in the control of the rate of oxaloacetate decarboxylation.  相似文献   

15.
Summary 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.Abbreviations AFR ascorbate free radical - DQ duroquinone - OA oxaloacetate - DPIP dichlorophenolindophenol - MDH malate dehydrogenase - PHMB p-hydroxymercuribenzoate  相似文献   

16.
We report herein the complete coding sequence of a Taenia solium cytosolic malate dehydrogenase (TscMDH). The cDNA fragment, identified from the T. solium genome project database, encodes a protein of 332 amino acid residues with an estimated molecular weight of 36517 Da. For recombinant expression, the full length coding sequence was cloned into pET23a. After successful expression and enzyme purification, isoelectrofocusing gel electrophoresis allowed to confirm the calculated pI value at 8.1, as deduced from the amino acid sequence. The recombinant protein (r-TscMDH) showed MDH activity of 409 U/mg in the reduction of oxaloacetate, with neither lactate dehydrogenase activity nor NADPH selectivity. Optimum pH for enzyme activity was 7.6 for oxaloacetate reduction and 9.6 for malate oxidation. Kcat values for oxaloacetate, malate, NAD, and NADH were 665, 47, 385, and 962 s−1, respectively. Additionally, a partial characterization of TsMDH gene structure after analysis of a 1.56 Kb genomic contig assembly is also reported.  相似文献   

17.
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.  相似文献   

18.
The trematodes Eurytrema pancreaticum and Calicophoron ijimai during the incubation in vitro assimilated glucose from the incubation medium and utilized the endogenous glycogen. Final products of the carbohydrate metabolism in the calicophorones were lactic, acatic, propionic, isobutyric and alpha-methylbutyric acids; in the eurytremes they were lactic, acetic, propionic, isobutyric, alpha-methylbutyric, valerianic and capronic acids. The effect of anthelminthic preparations on the carbohydrate metabolism and its final products was investigated.  相似文献   

19.
Low concentrations (less than 0.2% w/v) of phenoxyethanol stimulated both the rate of respiration and total oxygen uptakes of Escherichia coli NCTC 5933 suspensions with glucose and other substrates, whilst higher concentrations (0.2--0.6% w/v) although still below those showing significant bactericidal activity, produced progressive levels of inhibition. The degree of respiratory inhibition varied with different substrates in the order malate less than succinate less than pyruvate less than or equal to glucose less than lactate, and suggested appreciable inhibition at a point after malate in the tricarboxylic acid cycle. This suggestion was supported by the use of tetrazolium salts as alternative electron acceptors, and by cytochrome difference spectra, which together implicated malate dehydrogenase as the most likely site of action. Isolated dehydrogenase enzymes of the tricarboxylic acid cycle in cell-free preparations were unaffected by high concentrations of phenoxyethanol (0.8% w/v) with the exception of malate dehydrogenase which was inhibited in extracts to extents similar to those of malate oxidation by intact bacteria. Lineweaver-Burke plots for malate dehydrogenase activity in the presence of phenoxyethanol suggested a competitive inhibition of the oxaloacetic acid-limited reaction and a non-competitive inhibition of the NADH-limited reaction. Accordingly, Ki values were found to be low when the rate of reaction was limited by oxaloacetic acid concentration yet relatively high when NADH was rate limiting.  相似文献   

20.
To study the effect of facilitated diffusion of the intermediate metabolite, oxaloacetate, on the coupled reaction of aspartate aminotransferase (L-aspartate: 2-oxoglutarate aminotransferase, EC 2.6.1.1) and malate dehydrogenase (L-malate:NAD+ oxidoreductase, EC 1.1.1.37), these enzymes were co-immobilized on the surface of a collagen film. The kinetic properties of the immobilized enzymes were compared with those observed with the enzymes in solution. Since the reactions correspond to the cytosolic enzymes, they have been studied in the direction aspartate aminotransferase toward malate dehydrogenase. Coupled enzymes in solution showed classical behaviour. A lag-time was observed before they reached a steady state and this lag-time was dependent on the kinetic properties of the second enzyme, malate dehydrogenase. The same lag-time was observed when malate dehydrogenase in solution was coupled with aspartate aminotransferase bound to the film. When aspartate aminotransferase in solution was coupled with malate dehydrogenase bound to the collagen film, a very long lag-time was observed. Theoretical considerations showed that in the latter case, the lag-time was dependent on the kinetic properties of the second enzyme and the transport coefficient of the intermediate substrate through the boundary layer near the surface of the film. Then both enzymes were co-immobilized on the collagen film. The coupled activity of aspartate aminotransferase and malate dehydrogenase was compared for films with an activity ratio of 5 and 0.8. In both cases, a highly efficient coupling was observed. In the former case, where malate dehydrogenase was rate-limiting, 81% of this limiting activity was observed. In the latter case, aspartate aminotransferase was rate-limiting and 82% of its rate was obtained for the final product formation. The linear increase of product formation with time corresponded fairly well to the theoretical equations developed in the paper. To interpret these rate equations, one should assume that the intermediate substrate oxaloacetate formed by aspartate aminotransferase was used by malate dehydrogenase in the diffusion layer near the film, before diffusing in the bulk solution.  相似文献   

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