Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Changes in the metabolism of Crithidia fasciculata ATCC 11745 when grown in the presence of ethidium bromide were studied. Ethidium bromide-grown cells had decreased respiratory activity as measured by oxygen consumption. More than 50% of the organisms cultivated in a defined medium containing 1.0 mg/liter of ethidium bromide became dyskinetoplastic and had decreased activities of particulate succinate and NADH-linked dehydrogenases as well as of soluble isocitrate dehydrogenase. These cells also had increased activities of particulate alpha-glycerophosphate dehydrogenase, soluble alpha-glycerophosphate dehydrogenase, malic enzyme, hexokinase, and malate dehydrogenase. Ethidium bromide-grown cells had a lower level of ATP and contained less DNA than cells grown in its absence.
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PMID:Effect of ethidium bromide on the oxidative metabolism and enzyme profiles of Crithidia fasciculata. 86 23

2-(4-Bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-bisphosphate (2-BDB-T epsilon A-2',5'-DP) is an affinity label for the coenzyme-binding site of pig heart NADP+-dependent isocitrate dehydrogenase. Specific reaction occurs at the coenzyme site with an incorporation of 0.5 mol of reagent/mol of enzyme subunit (i.e. modification of only one subunit of the dimeric enzyme) (Bailey, J.M., and Colman, R.F. (1985) Biochemistry 24, 5367-5377). Modified enzyme, prepared by incubating 1 mg/ml isocitrate dehydrogenase with 75 microM 2-BDB-T epsilon A-2',5'-DP in the absence and presence of substrate or coenzyme, was reduced with NaBH4, carboxymethylated, and digested with trypsin. Nucleotidyl peptides were isolated by chromatography on DEAE-cellulose, followed by treatment with acid phosphatase (to decrease the negative charge by removing the phosphate groups from covalently bound reagent) and rechromatography on the same DEAE-cellulose column. The isolated peptides were characterized by amino acid analysis, dansylation, and gas-phase sequencing. A single triskaidekapeptide corresponding to modification of the coenzyme site by 2-BDB-T epsilon A-2',5'-DP was identified as: Asp-Leu-Ala-Gly-X-Ile-His-Gly-Leu-Ser-Asn-Val-Lys. Additional evidence indicated that X is a glutamate residue derivatized by 2-BDB-T epsilon A-2',5'-DP.
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PMID:Isolation of the glutamyl peptide labeled by the nucleotide analogue 2-(4-bromo-2,3-dioxobutylthio)-1,N(6)-ethenoadenosine 2',5'-biphosphate in the active site of NADP+-specific isocitrate dehydrogenase. 288 70

Two new reactive adenine nucleotide analogues have been synthesized and characterized: 2-[(4-bromo-2,3-dioxobutyl)thio]adenosine 2',5'-bisphosphate (2-BDB-TA-2',5'-DP) and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2',5'-bisphosphate (2-BOP-TA-2',5'-DP). Starting with NADP+, 2'-phospho-adenosine 5'-(diphosphoribose) (PADPR) was generated enzymatically and was converted to PADPR 1-oxide by reaction with m-chloroperoxybenzoic acid. Treatment with NaOH followed by reaction with carbon disulfide yielded 2-thioadenosine 2',5'-bisphosphate (TA-2',5'-DP). Condensation of TA-2',5'-DP with 1,4-dibromobutanedione or 1,3-dibromo-2-propanone gave the final products 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP, respectively. The structure of these new reagents was determined by UV, 1H NMR, 31P NMR, and 13C NMR spectroscopy as well as by bromide and phosphorus analysis. Both of these reagents exhibit properties expected for an affinity label of the coenzyme site of NADP+-dependent isocitrate dehydrogenase. With both reagents, biphasic kinetics of inactivation are observed that can be described in terms of a fast initial phase of inactivation resulting in partially active enzyme of 6-7% residual activity, followed by a slower phase leading to total inactivation. The inactivation rate constants for both reagents exhibit a nonlinear dependence on reagent concentration, consistent with the formation of a reversible complex with the enzyme prior to irreversible modification. The enzyme incorporates both reagents to a limited extent and is protected against inactivation by NADP+ and NADPH. The reaction of these new nucleotide analogues with isocitrate dehydrogenase is compared to the much slower inactivation caused by bromoacetone, indicating the importance of the nucleotide moiety in the functioning of the affinity labels. It is likely that 2-BDB-TA-2',5'-DP and 2-BOP-TA-2',5'-DP will have general applicability as affinity labels for other NADP+ binding enzymes.
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PMID:2-[(4-Bromo-2,3-dioxobutyl)thio]- and 2-[(3-bromo-2-oxopropyl)thio]adenosine 2'5'-bisphosphate: new nucleotide analogues that act as affinity labels of nicotinamide adenine dinucleotide phosphate specific isocitrate dehydrogenase. 342 47

Two new adenine nucleotide analogues have been synthesized and characterized: 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate and 5'-diphosphate. The bromoketo and dioxobutyl moieties have the ability to react with the nucleophilic side chains of several amino acids, as well as with arginine. 6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate reacts irreversibly with rabbit muscle pyruvate kinase, causing inactivation. Addition of ADP to the reaction mixture (in the presence of Mg2+) markedly decreases the rate of inactivation. Pig heart NAD-dependent isocitrate dehydrogenase is allosterically activated by ADP, which reduces the Km for isocitrate. 6-[(4-Bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-diphosphate reacts irreversibly with isocitrate dehydrogenase, causing, rapidly, a loss of the ability of ADP to increase the initial velocity of assays conducted at low isocitrate concentrations and, more slowly, inactivation. Addition of ADP to the reaction mixture (in the presence of Mn2+) protects this enzyme against the loss of allosteric activation. It is proposed that the 6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenine nucleotides react at the active site of pyruvate kinase and at the ADP activating site of isocitrate dehydrogenase and that these compounds may have general applicability as affinity labels of catalytic and regulatory adenine nucleotide sites in proteins.
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PMID:6-[(4-bromo-2,3-dioxobutyl)thio]-6-deaminoadenosine 5'-monophosphate and 5'-diphosphate: new affinity labels for purine nucleotide sites in proteins. 646 41

DPN-dependent isocitrate dehydrogenase is composed of three distinct types of subunits: alpha, beta, and gamma which have molecular weights of about 40,000 but differ in isoelectric points. The relationship of subunit diversity to function was probed by use of chemical modification. 3-Bromo-2-ketoglutarate, a substrate and affinity label for the active site of isocitrate dehydrogenase, was shown to cause significant modification of all types of subunits. The substrate affinity label, 3-ene-2-keto-glutarate, labels each of the subunits equally. Approximately equal labeling of subunits was also found upon modification by cyanate of an essential lysyl residue in the isocitrate binding site. When enzyme was inactivated by a carbodiimide in the presence of glycine ethyl ester, both glutamate and aspartate residues reacted, and labeling of each type of subunit occurred. These studies suggest that the structurally distinct subunits of DPN-dependent isocitrate dehydrogenase are functionally similar and each type of subunit contains a substrate binding site.
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PMID:Chemical modification A probe of the structure and function of the subunits of DPN-dependent isocitrate dehydrogenase. 711 8

Cytosolic NADP(+)-dependent isocitrate dehydrogenase was purified to homogeneity from superovulated rat ovaries. Amino acid sequence information was obtained by analyzing peptides generated by digestion with either cyanogen bromide or trypsin. Eleven peptides were sequenced and a total of 146 amino acids were identified. Nine of these peptides were found to be 60-100% identical with sequences from mitochondrial NADP(+)-dependent isocitrate dehydrogenase. Conservation of amino acids was observed for residues that were previously identified as potentially binding isocitrate-Mg2+. Circular dichroism measurements showed that the structure is composed of approximately 35% alpha-helix and 21% beta-sheet segments. Temperature denaturation studies indicated that the enzyme is more stable in the presence of isocitrate.
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PMID:Structural characterization of cytosolic NADP(+)-dependent isocitrate dehydrogenase from rat ovary. 778 68

8-(4-Bromo-2,3-dioxobutylthio)nicotinamide adenine dinucleotide (8-BDB-TNAD), a new reactive NAD analog, was synthesized by coupling 8-thio-AMP with NMN, followed by condensation with 1,4-dibromobutanedione. Incubation of 160 microM 8-BDB-TNAD with the allosteric pig heart NAD-dependent isocitrate dehydrogenase causes time-dependent inactivation to a limit of 25% residual activity concomitant with incorporation of approximately 1 mol reagent/mol average subunit. In addition to binding sites for NAD and NADH, this enzyme has been shown to have regulatory sites for NADPH and for ADP (R. S. Ehrlich and R. F. Colman 1982, J. Biol. Chem. 257, 4769-4774). Marked protection against enzyme inactivation by 8-BDB-TNAD and incorporation is provided by the regulatory nucleotides NADPH or ADP, while NAD and NADH are less effective. The rate constant for inactivation shows a nonlinear dependence on 8-BDB-TNAD concentration which can be ascribed to reversible formation of an enzyme-reagent complex (KI = 83 microM) prior to an irreversible reaction (kmax = 0.0625 min-1). Analysis of the kinetic properties and binding characteristics of modified enzyme indicates that this enzyme retains the ability to bind ADP, but does not bind NADPH. Thus, 8-BDB-TNAD reacts at or near the allosteric NADPH site of pig heart NAD-dependent isocitrate dehydrogenase.
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PMID:8-(4-Bromo-2,3-dioxobutylthio)NAD: a new affinity label for NAD-specific isocitrate dehydrogenase. 810 65

The genes coding for two structurally different isocitrate dehydrogenase isozymes (IDH-I and IDH-II) of a psychrophilic bacterium, Vibrio sp. strain ABE-1, were cloned and sequenced. Open reading frames of the genes (icdI and icdII) are 1,248 and 2,229 bp in length, respectively. The amino acid sequences predicted from the open reading frames of icdI and icdII corresponded to the N-terminal amino acid sequences of the purified IDH-I and IDH-II, respectively. No homology was found between the deduced amino acid sequences of the isozymes; however, the IDH-I, a dimeric enzyme, had a high amino acid sequence identity (74.3%) to the Escherichia coli IDH. The deduced amino acid sequence of the IDH-II, a monomeric enzyme, was not related to any known sequence. However, the IDH-II had an amino acid sequence homologous to that of a cyanogen bromide-cleaved peptide containing a putative active-site methionyl residue of the monomeric IDH of Azotobacter vinelandii. The two genes (icdlI and icdII) were found to be tandemly located in the same orientation. Northern (RNA) blot analyses showed that the two genes are transcribed independently. Primer extension experiments located single transcriptional start sites 39 and 96 bp upstream of the start codons of icdI and icdII, respectively. The amount of icdI transcript but not icdII increased when Vibrio sp. strain ABE-1 cells were cultured in acetate minimal medium.
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PMID:Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. 822 30

The ability of Klebsiella pneumoniae (NCTC, CL687/80) to produce and, in turn, excrete glutamate has been equated with the presence of a large indigenous plasmid with an apparent molecular mass in the region of 96 +/- 2 kbp (n = 6). Unlike mitomycin C, novobiocine and ethidium bromide (curing agents), the use of sodium dodecyl sulphate (SDS) proved very effective in curing the plasmid with a relatively high frequency (6.25 x 10(-4)). Furthermore, the absence of isocitrate dehydrogenase (ICDH) activity in the cured strain strongly suggests that the structural gene encoding ICDH in this organism, in sharp contrast to all known ICDHs, is plasmid-encoded. Moreover, the SDS-based protocol reported for the isolation of the K. pneumoniae indigenous plasmid has proven successful with other organisms including Pseudomonad and Corynebacteria, as well as in recombinant strains of Escherichia coli.
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PMID:Isolation and curing of the Klebsiella pneumoniae large indigenous plasmid using sodium dodecyl sulphate. 1086 47

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