A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.
Evidence
2:
Inferred from Mutant PhenotypeUniProtKB
Three cases of uncommon childhood hematologic disorders are reported. At presentation, one patient had refractory anemia with an excess of blasts (RAEB) with partial 7-monosomy and was reclassified into RAEB "in transformation" thereafter. Another case was diagnosed as acute myelogenous leukemia with complete 7-monosomy. The other case was diagnosed as RAEB "in transformation" without chromosome aberrations. The cytogenetic studies of the patients with 7-monosomy revealed abnormal karyotypes on bone marrow cells, but normal karyotypes on peripheral blood cells. Polymorphonuclear cells from the two patients with 7-monosomy revealed reduced mitochondrial malate dehydrogenase activity, but those from the patient with RAEB "in transformation" without chromosome aberrations did not. Cytoplasmic malate dehydrogenase activity, having been defined as located on chromosome 2, was within the normal range in those three patients. The decreased mitochondrial enzyme activity in the two patients with 7-monosomy would be a dosage effect of the chromosome aberration, but not caused by their hematologic disorders. The level of mitochondrial enzyme activity in the patients with 7-monosomy was reduced in polymorphonuclear cells, but not in mononuclear cells in peripheral blood. This fact would indicate that such chromosome evolution had involved myeloid cells only, but not lymphoid cells. Both enzymes from leukemic cells of four patients with active disease revealed much higher activities than controls, an expression of partially enhanced oxidative phosphorylation.
Interacting selectively and non-covalently with a nucleotide, any compound consisting of a nucleoside that is esterified with (ortho)phosphate or an oligophosphate at any hydroxyl group on the ribose or deoxyribose.
The chemical reactions and pathways involving carbohydrates, any of a group of organic compounds based of the general formula Cx(H2O)y, as carried out by individual cells.
Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.
The chemical reactions and pathways involving malate, the anion of hydroxybutanedioic acid, a chiral hydroxydicarboxylic acid. The (+) enantiomer is an important intermediate in metabolism as a component of both the TCA cycle and the glyoxylate cycle.
Evidence
1:
Inferred from Mutant PhenotypeUniProtKB
Three cases of uncommon childhood hematologic disorders are reported. At presentation, one patient had refractory anemia with an excess of blasts (RAEB) with partial 7-monosomy and was reclassified into RAEB "in transformation" thereafter. Another case was diagnosed as acute myelogenous leukemia with complete 7-monosomy. The other case was diagnosed as RAEB "in transformation" without chromosome aberrations. The cytogenetic studies of the patients with 7-monosomy revealed abnormal karyotypes on bone marrow cells, but normal karyotypes on peripheral blood cells. Polymorphonuclear cells from the two patients with 7-monosomy revealed reduced mitochondrial malate dehydrogenase activity, but those from the patient with RAEB "in transformation" without chromosome aberrations did not. Cytoplasmic malate dehydrogenase activity, having been defined as located on chromosome 2, was within the normal range in those three patients. The decreased mitochondrial enzyme activity in the two patients with 7-monosomy would be a dosage effect of the chromosome aberration, but not caused by their hematologic disorders. The level of mitochondrial enzyme activity in the patients with 7-monosomy was reduced in polymorphonuclear cells, but not in mononuclear cells in peripheral blood. This fact would indicate that such chromosome evolution had involved myeloid cells only, but not lymphoid cells. Both enzymes from leukemic cells of four patients with active disease revealed much higher activities than controls, an expression of partially enhanced oxidative phosphorylation.
A sensitive spectrophotometric assay for determining mitochondrial malate dehydrogenase activity is described. The assay measures NADH production by coupling it to the reduction of 2-(p-iodophenyl)-3(p-nitrophenyl)-5-phenyl tetrazolium chloride (INT). Via an intermediate electron carrier, either phenazine methosulfate or lipoamide dehydrogenase, INT accepts electrons and is reduced to a red-colored formazan, which can be quantified by spectrophotometer at 500 nm. This assay uses only commercial reagents but gives a 2-5 fold (with lipoamide dehydrogenase) or 5-20 fold (with phenazine methosulfate) activity increase over currently available assays for pure enzyme in mitochondria isolated from human neuroblastoma cells, rat brain and liver, and crude homogenates of rat brain and liver. The assay can be easily performed with 96-well plate and less than 2.5 microg protein of isolated mitochondria or crude tissue homogenate. These results suggest that this assay is a simple, sensitive, stable and inexpensive method with wide application.
The chemical reactions and pathways involving reduced nicotinamide adenine dinucleotide (NADH), a coenzyme present in most living cells and derived from the B vitamin nicotinic acid.
The chemical reactions and pathways involving oxaloacetate, the anion of oxobutanedioic acid, an important intermediate in metabolism, especially as a component of the TCA cycle.
IEAOrtholog Compara
Enzymatic activity
This protein acts as an enzyme. It is known to catalyze the following reaction
Protein lysine acetylation has emerged as a key posttranslational modification in cellular regulation, in particular through the modification of histones and nuclear transcription regulators. We show that lysine acetylation is a prevalent modification in enzymes that catalyze intermediate metabolism. Virtually every enzyme in glycolysis, gluconeogenesis, the tricarboxylic acid (TCA) cycle, the urea cycle, fatty acid metabolism, and glycogen metabolism was found to be acetylated in human liver tissue. The concentration of metabolic fuels, such as glucose, amino acids, and fatty acids, influenced the acetylation status of metabolic enzymes. Acetylation activated enoyl-coenzyme A hydratase/3-hydroxyacyl-coenzyme A dehydrogenase in fatty acid oxidation and malate dehydrogenase in the TCA cycle, inhibited argininosuccinate lyase in the urea cycle, and destabilized phosphoenolpyruvate carboxykinase in gluconeogenesis. Our study reveals that acetylation plays a major role in metabolic regulation.
Protein involved in the tricarboxylic acid cycle, a series of metabolic reactions in aerobic cellular respiration, which occurs in the mitochondria of animals and plants and in which acetyl-CoA, formed from pyruvate produced during glycolysis, is completely oxidized to CO2 via the interconversion of various carboxylic acids. It results in the reduction of NAD and FAD to NADH and FADH2, whose reducing power is then used indirectly in the synthesis of ATP by oxidative phosphorylation. The TCA cycle also provides intermediates for many other biosynthetic processes.
A reference proteome is a set of protein sequences derived from a complete proteome which constitutes a defined standard for a particular user community. Reference proteomes are manually defined according to a number of criteria. They cover the proteomes of well- studied model organisms and other proteomes of interest for biomedical and biotechnological research. Reference proteomes have been selected to provide broad coverage of the tree of life, and constitute a representative cross-section of the taxonomic diversity to be found within UniProtKB.
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