Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:1.1.1.41 (isocitrate dehydrogenase)
3,101 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The interaction of oxidized nicotine adenine dinucleotide phosphate dependent isocitrate dehydrogenase (from pig heart) with (RS)-3-bromo-2-ketoglutarate was investigated in an effort to evaluate the reagent's potential as a selective reagent for alpha-ketoglutarate binding sites. The enzyme is rapidly inactivated by 0.1 mM bromoketoglutarate at pH 7.4. With increasing concentrations of regent, the reaction shows a rate saturation; the minimum inactivation half-time is 3 min and Kinact for bromoketoglutarate is 250 microM. Isocitrate and NADP+ protect against inactivation, while ketoglutarate does not. When tested in the assay that monitors isocitrate oxidation, bromoketoglutarate is a competitive inhibitor (Ki = 100 microM) of the dehydrogenase. As judged by oxidation of NADPH, bromoketoglutarate is also a substrate for isocitrate dehydrogenase, exhibiting a Km of 250 microM and a Vmax comparable to that for isocitrate oxidation. The reduction of bromoketoglutarate is competitively inhibited by isocitrate (Ki = 3 microM) and ketoglutarate (Ki = 50 microM). Like the enzyme-catalyzed oxidation of isocitrate, the reduction of bromoketoglutarate is stereospecific, requires divalent metal ions, and shows absolute specificity for NADPH. However, since CO2 is not required for catalytic turnover of bromoketoglutarate, its reduction is likely comparable to that of oxalosuccinate rather than the reductive carboxylation of ketoglutarate. Although bromoketoglutarate, as a substrate for isocitrate dehydrogenase, clearly has affinity for the active site, the irreversible inactivation of the enzyme by the reagent may result from modification outside the active-site region, since inactivation during catalytic turnover of bromoketoglutarate is not observed. Commercial isocitrate dehydrogenase is purified 12-fold by affinity chromatography on thiol-agarose alkylated by bromoketoglutarate.
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PMID:Interaction of isocitrate dehydrogenase with (RS)-3-bromo-2-ketoglutarate. A potential affinity label for alpha-ketoglutarate binding sites. 721 20

Neurite outgrowth from explants of superior cervical ganglion from adult rats can be achieved in a serum-free medium. Extensive neurite outgrowth occurred from ganglion explants maintained in Eagle's minimum essential medium supplemented with either 10% (V/V) fetal calf serum or 1% (W/V) bovine serum albumin and nerve growth factor. After one week in culture, the ATP content of explants maintained in the serum-free medium was slightly higher than that noted in explants cultured in the presence of fetal calf serum and amounts of phosphocreatine were significantly lower. Despite these differences in high energy phosphate content, the abundance and morphology of neuritic outgrowth were essentially the same from explants cultured in the two types of media. Comparable activities of a number of NADP+-dependent dehydrogenases were noted in explants maintained in the two types of media. Increases in the activities of the oxidative enzymes of the pentose pathway, which occur in axotomized ganglia in vivo, were observed in the cultured ganglion explants. NADP+-dependent isocitrate dehydrogenase activity remained constant in ganglion explants in vitro, and measurements of this activity were employed in a new method to quantitate neurite outgrowth. The activity of isocitrate dehydrogenase in lyophilized neurite processes that had grown out onto a Millipore filter substrate correlated well with visual estimates of neuritic outgrowth. Substitution of delipidated for normal bovine serum albumin in the growth medium resulted in a significant decrease in neuritic outgrowth from ganglion explants from both adult and weanling rats. Addition of fatty acids to media containing delipidated bovine serum albumin enhanced neuritic outgrowth in explants of weanling rats. Thus, lipophilic substances bound to bovine serum albumin including fatty acids appear necessary for optimal growth of neurites from explants of the rat superior cervical ganglion.
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PMID:Growth of adult superior cervical ganglion explants in serum-free media. 726 Jun 37

The developmental and senescent patterns of a number of heart enzyme activities linked to energy metabolism have been studied in rats aged between 4 days and 21 months. A morphometric study of mitochondrial volume fractions and numbers has been also carried out. Developmental changes result in a rise of most mitochondrial enzymes (NADP+-isocitrate dehydrogenase, malic enzyme, succinate dehydrogenase, citrate synthase) and mitochondrial volume fractions. Exceptions are NAD+-isocitrate dehydrogenase, which declines from 4 days onwards, and NAD+-malate dehydrogenase, which declines and then rises over the same period. Senescent changes follow two different trends. While pyruvate kinase and those mitochondrial enzymes lying between citrate formation and isocitrate oxidation (citrate synthase, NADP+-and NAD+-isocitrate dehydrogenases) decline to some degree, mitochondrial succinate dehydrogenase and NAD+-malate dehydrogenase activities increase over the same period. This could point towards a partial impairment of Krebs cycle function, and a reduced energy-producing capacity in the aged rat heart.
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PMID:Comparison between developmental and senescent changes in enzyme activities linked to energy metabolism in rat heart. 726 74

In crude extract of castor bean endosperm, isocitrate dehydrogenase (NADP+) (EC 1.1.1.42) was stable at 57 degrees C at the beginning of seed germination as well as in maturing and dry seeds. The enzyme gradually became less thermostable as germination proceeded and became unstable after 4 days. Extract from 5-day-old endosperm reduced the thermostability of the thermostable enzyme. The destabilizing factor accumulated in the endosperm as germination progressed and was identified as ricinoleate. Ricinoleate destabilized the purified enzyme which was stabilized by isocitrate and Mg2+, but ricinoleate did not affect the activity of NADP+-isocitrate dehydrogenase itself. Stearate, oleate, palmitate and myristate were similar to ricinoleate in their effect on the thermostability of the enzyme. The thermolabile enzyme in the crude extract of 5-day-old endosperm was readily inactivated by trypsin and in low concentrations of buffer. The thermostable enzyme in the crude extract of 2-day-old endosperm was not affected by these treatments. The thermostable enzyme treated with ricinoleate showed the same instabilities as the thermolabile enzyme. The role of ricinoleate in the germinating castor bean endosperm is discussed.
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PMID:Changes in stability of isocitrate dehydrogenase (NADP+) during germination of castor bean seeds. 739 30

Cell-free extracts of the obligate methanol-utilizing bacterium Pseudomonas W6 catalyze the oxydation of isocitrate to alpha-ketoglutarate in the presence of NAD+ and NADP+. After electro-focusing of the crude extract of Pseudomonas W6 actually two distinct bands each of NAD+-linked isocitrate dehydrogenase (NAD+-IDH) and of NADP+-linked isocitrate dehydrogenase (NADP+-IDH) could be observed. The NAD+-IDH was completely separated from the NADP+-IDH by employing DEAE ion exchange chromatography and further purified by affinity chromatography using Cibacron blue F 3G-A. The NAD+-IDH was inhibited by a high energy charge, whereas the NADP+-IDH was found to be independent of energy charge. Consequently the NAD+-IDH showed the control behaviour of an enzyme of an energy-generating sequence which, however, equally fulfils a catabolic and an anabolic function. With respect to the inhibition by reduced pyridine nucleotides and alpha-ketoglutarate differences between NAD+-IDH and NADP+-IDH were also found. Only the NADP+-linked enzyme exhibited a feedback inhibition by its reaction products alpha-ketoglutarate and NADPH. This control behaviour gives evidence for the biosynthetic function of the NADP+-IDH. These results confer an amphibolic character to the sequence from citrate to alpha-ketoglutarate in the incomplete citric-acid cycle of Pseudomonas W6.
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PMID:Regulation of NAD+- and NADP+-linked isocitrate dehydrogenase in the obligate methylotrophic bacterium Pseudomonas W6. 742 52

Reactive coenzyme analogues omega-(3-diazoniumpyridinium)alkyl adenosine diphosphate were prepared by reaction of omega-(3-aminopyridinium)alkyl adenosine diphosphate with nitrous acid. In these compounds the nicotinamide ribose is substituted by hydrocarbon chains of varied lengths (n-ethyl to n-pentyl). The diazonium compounds are very unstable and decompose rapidly at room temperature. They show a better stability to 0 degree C. Lactate and alcohol dehydrogenase do not react with any of the analogues. Glyceraldehyde-3-phosphate dehydrogenase reacts rapidly with the diazoniumpentyl compound. Decreasing the length of the alkyl chain significantly decreases the inactivation velocity. 3 alpha, 20 beta-Hydroxysteroid dehydrogenase reacts at 0 degree C with the ethyl homologue and slowly with the propyl compound. The butyl- and pentyl analogues do not inactivate at 0 degree C. Tests with 14C-labeled 2-(3-diazoniumpyridinium)ethyl adenosine diphosphate show that complete loss of enzyme activity results after incorporation of 2 moles of inactivator into 1 mole of tetrameric enzyme. 4-(3-Acetylpyridinium)butyl 2'-phospho-adenosine diphosphate, a structural analogue of NADP+, was prepared by condensation of adenosine-2,3-cyclophospho-5'-phosphomorpholidate with (3-acetylpyridinium)butyl phosphate, followed by hydrolysis of the cyclic phosphoric acid with 2':3'-cyclonucleotide-3'-phosphodiesterase. Because of the redox potential (-315 mV) and the distance between the pyridinium and phosphate groups, this analogue is a hydrogen acceptor and its reduced form a hydrogen donor in tests with alcohol dehydrogenase from Thermoanaerobium brockii. The reduced form of the coenzyme analogue also is a hydrogen donor with glutathione reductase. With other NADP+-dependent dehydrogenases the compound has been shown to be a competitive inhibitor against the natural coenzyme. The acetyl group reacts with bromine to form the bromoacetyl group. This reactive bromoacetyl analogue is a specific active-site directed irreversible inhibitor of isocitrate dehydrogenase.
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PMID:New reactive coenzyme analogues for affinity labeling of NAD+ and NADP+ dependent dehydrogenases. 754 38

The cytosolic form of NADP+:isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by fatty acyl-coenzyme A (CoA). The reduction of NADP+ by the enzyme is inhibited by palmitoyl-CoA. In steady-state experiments, when added enzyme is used to start the reaction, analyses of velocity versus palmitoyl-CoA concentration as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, suggested that binding of palmitoyl-CoA produced two different inhibitory effects on the enzyme. This analysis suggested inhibition first through binding to sites with an average dissociation constant of 3.3 microM, then by binding to sites with an average dissociation constant of 294 microM. When the enzyme is preincubated with palmitoyl-CoA there is an induction of a significant lag-burst reaction rate (hysteretic kinetics). Preincubation of the enzyme with its substrate, metal-isocitrate complex, nearly abolished the lag time and decreased the degree of inhibition. Changes in lag time and percentage inhibition as a function of concentration of palmitoyl-CoA followed patterns, similar to those observed in steady-state reactions, where the enzyme is not preincubated. Examination of the effect of acyl chain length at 300 microM demonstrated that only long-chain CoA's with carbon numbers > 14 have pronounced effects on kinetics. CoA alone has little or no effect, while stearoyl-CoA completely inhibited the enzyme. Other C18 acyl groups produced varying effects depending on the degree of unsaturation and cis-trans isomerism. NADP+:Isocitrate dehydrogenases, from other sources including that from Escherichia coli, do not show such sensitivity to acyl chain character under these conditions. Concentration ranges observed for these transitions are compatible with physiological conditions. This suggests that complexes of acyl-CoA's and NADP+:isocitrate dehydrogenase, in tissue rich in the cytoplasmic form of the enzyme, could be related to cytoplasmic events in the synthesis and secretion of lipid and possibly protein, since palmitoyl-CoA is known to promote secretory processes through acylation reactions which lead to vesicle fusion.
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PMID:Effects of long-chain acyl-coenzyme A's on the activity of the soluble form of nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from lactating bovine mammary gland. 763 21

Bovine NAD(+)-dependent isocitrate dehydrogenase was shown previously to contain four subunits of approx. 40 kDa (subunits 1-4) possessing different peptide maps and electrophoretic properties [Rushbrook and Harvey (1978) Biochemistry 17, 5339-5346]. In this study the heterogeneity is confirmed using enzyme purified by updated methods and from single animals, ruling out allelic variability. Subunits 1 and 2 were differentiated from each other and from subunits 3 and 4 by N-terminal amino acid sequencing. Subunits 3 and 4 (subunits 3/4) were identical in sequence over 30 residues. The N-terminal residues of subunits 1 and 2 were homologous but not identical with the beta- and gamma-subunits respectively of the comparable pig heart enzyme. Subunits 3/4 were identical over 30 residues with the N-terminus of the pig heart alpha-subunit. Full-length sequence, including that for mitochondrial import, is presented for a protein with the processed N-terminus of subunits 3/4, deduced from cloned cDNA obtained utilizing the N-terminal sequence information. The derived amino acid sequence for the mature protein contains 339 amino acids and has a molecular mass of 36,685 Da. Complete identity with N-terminal and Cys-containing peptides totalling 92 residues from the alpha-subunit of the pig heart enzyme [Huang and Colman (1990) Biochemistry 29, 8266-8273] suggests that maintenance of a particular three-dimensional structure in this subunit is crucial to the function of the enzyme. An electrophoretic heterogeneity within the pig heart alpha-subunit, similar to that shown by bovine subunits 3/4, was demonstrated. One reordering of the Cys-containing peptides of the pig heart alpha-subunit is indicated. Sequence comparison with the distantly related NADP(+)-dependent enzyme from Escherichia coli, for which the three-dimensional structure is known [Stoddard, Dean and Koshland (1993) Biochemistry 32, 9310-9316] shows strong conservation of residues binding isocitrate, Mg2+ and the NAD+ moiety of NADP+, consistent with a catalytic function.
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PMID:Isocitrate dehydrogenase from bovine heart: primary structure of subunit 3/4. 765 89

We report a method for assaying magnesium in serum and urine involving only one enzyme, isocitrate dehydrogenase (NADP+)(EC 1.1.1.42), which requires magnesium ion for activity. The enzymatic reduction of NADP+ by isocitrate increases in rate linearly up to at least 20 mmol/L magnesium in the presence of appropriate concentrations of the two metal-chelating reagents, EDTA and glycol ether diamine-N,N,N',N'-tetraacetate. Within-run (n = 20) CVs and day-to-day (n = 10) CVs for sera are < or = 1.5% and < or = 2.6%, respectively. Analytical recovery of magnesium in sera averages 96-100%. This method is not affected by bilirubin, hemoglobin, or lipemia. The method (y) gives the following results correlating with atomic absorption spectrophotometry (x): y = 1.03x + 0.06 mmol/L (n = 62, r = 0.995, Sylx = 0.03) for sera, and y = 1.03x - 0.10 mmol/L (n = 62, r = 0.989, Sylx = 0.19) for urines; with the calmagite method (x): y = 0.99x + 0.04 mmol/L (n = 62, r = 0.991, Sylx = 0.03) for sera, and y = 0.98x + 0.03 mmol/L (n = 62, r = 0.999, Sylx = 0.02) for urines.
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PMID:Assay of magnesium in serum and urine with use of only one enzyme, isocitrate dehydrogenase (NADP+). 765 41

NADP(+)-dependent isocitrate dehydrogenase (ICD) is an important enzyme of the intermediary metabolism, as it controls the carbon flux within the citric acid cycle and supplies the cell with 2-oxoglutarate and NADPH for biosynthetic purposes. In the amino acid-producing organism Corynebacterium glutamicum, the specific activity of ICD was independent of the growth substrate and of the growth phase at approximately 1 U/mg, indicating that this enzyme is constitutively formed. The ICD gene, icd, was isolated, subcloned on a plasmid, and introduced into C. glutamicum. Compared with the wild type, the recombinant strains showed up to 10-fold-higher specific ICD activities. The nucleotide sequence of a 3,595-bp DNA fragment containing the icd gene was determined. The predicted gene product of icd consists of 739 amino acids (M(r) = 80.091) and showed 58.5% identity with the monomeric ICD isozyme II from Vibrio sp. strain ABE-1 but no similarity to any known ICD of the dimeric type. Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity, suggesting that only a single ICD is present in C. glutamicum. From an icd-overexpressing C. glutamicum strain, ICD was purified and biochemically characterized. The native ICD was found to be a monomer; to be specific for NADP+; to be weakly inhibited by oxaloacetate, 2-oxoglutarate, and citrate; and to be severely inhibited by oxaloacetate plus glyoxylate. The data indicate that ICD from C. glutamicum is structurally similar to ICDs from bacteria of the genera Vibrio, Rhodomicrobium, and Azotobacter but different from all other known procaryotic and eucaryotic ICDs.
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PMID:Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. 783 12


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