Effect of Nitric Oxide on the Interaction Between Mitochondrial Malate Dehydrogenase and Citrate Synthase

Mitochondrial malate dehydrogenase （mMDH） and citrate synthase （CS） are sequential enzymes in Krebs cycle. mMDH, CS and the complex between mMDH and CS （mMDH＋CS） were treated with nitric oxide solution. The roles of notric oxide （NO） on the secondary structures and the interactions between mMDH and CS were studied using circular diehroism （CD） and Fourier transform surface plasmon resonance （FT-SPR）, respectivley. The effects of NO on the activities of mMDH, CS and mMDH＋CS were also studied. And the regulations by NO on mMDH and CS were simulated by PyMOL software. The results of SPR conifrmed that strong interaction between mMDH and CS existed and NO could signiifcantly regulate the interaction between the two enzymes. NO reduced the mass percents ofα-helix and increased that of random in mMDH, CS and mMDH＋CS. NO increased the activities of CS and mMDH＋CS, and inhibited the activity of mMDH. Graphic simulation indicated that covalent bond was formed between NO and Asn242 in active site of CS. However, there was no direct bond between NO and mMDH. The increase in activity of mMDH＋CS complex depended mostly on the interaction between NO and CS. All the results suggested that the regulations by NO on the activity and interaction between mMDH and CS were accord with the changes in mMDH, CS and mMDH＋CS caused by NO.

Molecular Weight of Cytoplasmic Malate Dehydrogenase,Mitochondrial Malate Dehydrogenase and Lactate Dehydrogenase of a Freshwater Catfish

The multiple molecular forms of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH ) and lactate dehydrogenase (LDH ) were studied in the liver and skeletal muscle of the freshwater catfish, Clarias batrachus. There were two electrophoretically distinguishable bands (AA andBB) of cMDH and mMDH which suggests that they are apparently encoded at two gene loci (A and B) in both the tissues.However, the presence of a single band (LDH-1 ) of LDH in liver and double bands (LDH-1and LDH-2) in skeletal muscle in which LDH-2 was predominant reflects the differential expression of LDH genes in different metabolic tissues to meet the requirement of energy production. The AA isoform (74 kd) of liver cMDH was smaller than those of the AA form (110 kd) of skeletal muscle. In contrast, the BB isoform of liver (42 kd) and skeletal muscle (54 kd) were more or less similar in size. Unlike the case of cMDH, the molecular weight of AA isoform (115 kd) of liver mMDH was higher than those of the AA form (87kd) of skeletal muscle. Whereas the molecular weight of BB isoform (58 kd) of liver was in proximity to the weight of BB form (44 kd) of skeletal muscle mMDH. The size of AA isoform (74 kd) of liver cMDH was smaller, while the AA isoform (110 kd) of skeletal muscle was larger as compared to AA form of mMDH in the liver (115 kd) and skeletal muscle (87 kd). But the size of BB isoform of both the isozymes was almost equal in these metabolic tissues. The molecular weight of liver LDH-1 (96 kd) was close to the weight of LDH-1 (82 kd) in skeletal muscle. The molecular weight of skeletal muscle LDH-2 was deduced as 37 kd which is much more lower than the weight of LDH-1 in liver and skeletal muscle. The smaller size of LDH-2 in skeletal muscle may be of a physiological significance in this anaerobic tissue

Duplication of Locus Coding of Malate Dehydrogenase in Populus tomentosa Carr

Horizontal starch-gel electrophoresis was used to study crude enzyme extraction from young leaves of 234 clones of Populus tomentosa Cart. selected from nine provenances in North China. Ten enzyme systems were resolved. One hundred and fifty-six clones showing unusual allozyme band patterns at locus Mdh-Ⅰ were found. Three allozyme bands at locus Mdh-Ⅰ were 9：6：1 in concentration. Further studies on the electrophoretic patterns of ground mixed pollen extraction of 30 male clones selected at random from the 156 clones were conducted and it was found that allozyme bands at locus Mdh-Ⅰ were composed of two dark-stained bands and a weak band. Only one group of the malate dehydrogenase （MDH） zymogram composed of two bands was obtained from the electrophoretic segregation of pollen leachate of the same clones. A comparison of the electrophoretic patterns one another suggested that the locus Mdh-Ⅰ coding malate dehydrogenase in diploid species of P. tomentosa was duplicated. The duplicate gene locus possessed three same alleles and was located in mitochondria. The locus duplication of alleles coding malate dehydrogenase in P.tomentosa was discovered and reported for the first time.

Molecular Characterization of a Cytosolic Malate Dehydrogenase Gene（GhcMDH1） from Cotton

Malate dehydrogenase（MDH） is a key enzyme that catalyzes the reversible oxidation of oxaloacetate to malate and plays an important role in the physiological processes of plant growth and development. However, cyto- solic malate dehydrogenase（cMDH）, which is crucial for malate synthesis in the cytosol, still has not been extensively characterized in plants. Here, we isolated a cytosolic malate dehydrogenase gene, designated as GhcMDH1, from Gossypium hirsutum and characterized its possible molecular function in cotton fiber. The cloned cDNA of GhcMDH1 is 1520 base pairs in length, and has an open reading frame of 999 base pairs, encoding for 332 amino acid residues with an estimated molecular weight of 35580 and pI of 6.35. Sequence alignment showed that the de- duced amino acid sequence of GhcMDH1 protein shared a high similarity to other plant cMDHs. Confocal and im- munological analysis confirmed that GhcMDH1 protein was subcellularly localized to the cytosol. Quantitative real-time polymerase chain reaction（PCR） revealed that GhcMDH1 was constitutively expressed in all vegetative cotton tissues, with slightly lower levels in roots than stems and leaves. Interestingly, the transcripts of GhcMDH1 were detected in 5--25 d post anthesis（DPA） fibers and highly abundant at 15 DPA fibers. The total MDH activities and malate contents of cotton fibers were positively correlated with the fiber elongation rates, suggesting that GhcMDH1 may function in malate synthesis in fast fiber elongation. In agreement with this suspicion, the recombi- nant His-GhcMDH1 protein mainly drives the reaction towards malate generation in vitro. In conclusion, our mole- cular characterization of the GhcMDH1 gene provides valuable insights to further investigate the malate equilibrium in cotton fiber development.

Malate and Lactate Dehydrogenases of a Freshwater Catfish: Impact of Endosulfan

A sublethal concentration of technical grade endosulfan (END) inhibited 35 to 55% of the activities of cytoplasmic malate dehydrogenase (cMDH), mitochondrial malate dehydrogenase (mMDH), and lactate dehydrogenase (LDH) in the liver and the skeletal muscle of a freshwater catfish, Clarias halrachus, after 7 days of exposure. The activity remained in the inhibited state up to 28 days. The withdrawal of END from the medium after 1 week of exposure gradually restored the activities to control levels within 21 days in the skeletal muscle and 28 days in the liver. The administration of actinomycin D or cycloheximide between the 14th and the 21st day of the withdrawal of END almost completely inhibited the withdrawal-dependent recovery in the activities of all the three enzymes. This indicates de novo synthesis of the enzymes during the recovery period. A conjoint treatment of END and triiodothyronine (T_3) raised the activities of cMDH, mMDH, and LDH in the liver and the skeletal muscle up to the control levels. This shows that the inhibitory effect of END may be relieved in presence of T_3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed few changes in the pattern of cytoplasmic proteins of the liver and the skeletal muscle in response to exposure to END. 1990 Academic Press. Inc.

A Review on Molecular Physiology of Malate and Lactate Dehydrogenases in Fishes

The malate(EC 1.1.1.37)and lactate(EC 1.1.1.27)dehydrogenases are themetabolic enzymes directly or indirectly involved in energy production,gluconeogenesis and lipogenesis.Malate dehydrogenase(MDH)exists in twoisoenzymic forms,cytoplasmic(cMDH)and mitochondrial(mMDH),composed of Aand/or B subunits(dimeric molecule:MW 40,000-120,000).Lactate dehydrogenase(LDH)has tetrameric(MW 35,000-110,000)structure made up of either A and/or B,orC(C,E,F)subunits.They catalyze an ordered bisubstrate(substrate and coenzyme)

Comparative Proteomic Analysis Shows an Elevation of Mdh1 Associated with Hepatotoxicity Induced by Copper Nanoparticle in Rats

Copper nanoparticle is a new material widely used in biological medicine, animal husbandry and industrial areas, but its potential toxicity to human health and environment remains unclear. In order to study the hepatotoxic mechanisms of nanoparticles copper, two-dimensional gel electrophoresis （2-DE） and matrix-assisted laser desorption/ionization time of flight mass spectrometry （MALDI/TOF MS） of proteomics technology were used to isolate and identify the differentially expressed proteins from liver, which associated with hepatotoxicity induced by copper nanoparticle in rats. In this study, we have screened 15 kinds of proteins related with hepatotoxicity, of which spot8212 was identified as Malate dehydrogenase （Mdhl）. The mRNA expression trend of Mdhl was consistent with the result of 2-DE by RT-PCR validation. Bioinformatics analysis showed that Mdhl was stable and no signal peptides, subcellular location was in endoplasmic reticulum; it contained many functional sites such as malate dehydrogenase activity signal sites 155LTRLDHNRAKSQI167; a helixes and random coils were the two main elements. Homologous analysis demonstrated high homologous of Mdhl in rats with mouse and human, and the phylogenetic tree of Mdhl was constructed. The result indicated that copper nanoparticle could regulate up the Mdhl protein expression so as to compensate the energy deficit. Energy metabolic disturbance may be a pathway for copper nanoparticle particles to exert the hepatotoxic effects in rats.