Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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Glucose-6-phosphate dehydrogenase was purified to homogeneity from testes and kidneys of the inbred strain of mice (DBA/2J) by a simple two-step affinity column procedure. This involved the sequential application of 8-(6-aminohexyl)-amino-AMP- and -2', 5'-ADP-Sepharose columns and biospecific elution with NADP+ in both steps. The molecular and biochemical properties of the purified enzyme were studied in detail. These include the molecular weight determination, amino acid composition, steady-state kinetics, inactivation by high temperature, urea and iodoacetate, and immunology. The purified enzyme from mouse kidneys or testes was shown to be a tetramer with a molecular weight of 220,000. The enzyme is highly specific for glucose-6-phosphate, exhibits almost no activity with NAD+ as a coenzyme and is little inhibited by AMP or ATP. Michaelis constants for glucose-6-phosphate and NADP+ were determined to be 50 microM and 10 microM respectively. NADPH is a competitive inhibitor of NADP+ and has a Ki of 18 microM. Rabbit antisera against glucose-6-phosphate dehydrogenase were raised. The antisera also cross-react with the same enzyme from human and guinea pig.
Mol Cell Biochem 1979 Mar 19
PMID:Purification and characterization of mouse glucose 6-phosphate dehydrogenase. 46 Jan 73

Cytoplasmic and mitochondrial isozymes of NADP+-dependent isocitrate dehydrogenase were purified from kidney and heart tissue of an inbred strain of mice. The cytoplasmic isozyme was purified from kidney of DBA/2J mice by means of a four-step procedure which included affinity chromatography with an 8-(6-aminohexyl)-amino-NADP+-Sepharose column. The heart mitochondrial isozyme of DBA/2J mice was purified by a two-step procedure involving the use of 8-(6-aminohexyl)-amino-AMP-Sepharose and 8-(6-aminohexyl)-amino-NADP+-Sepharose columns. The specific activity of the homogeneous cytoplasmic and mitochondrial isozymes was 40 units/mg and 45 units/mg, respectively. Native and subunit molecular weights of these two isozymes were determined by chromatography on Sephadex G-100, G-150 and G-200 Superfine and polyacrylamide gel electrophoresis. Both isozymes were found to be dimers with the subunit molecular weight of approximately 35,000. The sedimentation coefficients were determined to be 5.9 and 6.1 for the mitochondrial and cytoplasmic isozyme, respectively. The amino acid compositions of these two isozymes revealed distinct differences in arginine and proline contents. A modified procedure regarding the use of affinity columns for the purification of the weakly bound enzymes is also discussed.
Mol Cell Biochem 1979 Feb 09
PMID:Purification and structural properties of isozymes of isocitrate dehydrogenase from the mouse. 48 28

Transplantable rat liver tumors 5123 t.c., 7288 ct.c., 5123 t.c.(H) and the Novikoff hepatoma have active mixed function oxidase systems capable of metabolizing a variety of drug and polycyclic hydrocarbon substrates. The tumor drug metabolism systems are at best 20% as active as rat liver. The tumor drug metabolism activities are induced by pretreatment with phenobarbital or beta-naphthoflavone and can be inhibited with specific inhibitors such as carbon monoxide or 7,8-benzoflavone. Tumor drug metabolism systems appear to consist of cytochrome P-450 and cytochrome P-450 reductase. The properties of the two protein components from tumors are highly similar to the corresponding components of the liver drug metabolism system. Cytochrome P-450 reductase has been at least partially purified from the Novikoff hepatoma and hepatoma 5123 t.c.(H). The kinetic and physical properties of the tumor reductases are similar to those of the liver reductase except that the Km of hepatoma 5123 t.c.(H) reductase, but not of the Novikoff hepatoma reductase for NADPH, is elevated an order of magnitude over the Km of the liver reductase. The mechanism for the interaction of electron donor and electron acceptor with liver or tumor reductases seems to be a sequential reaction mechanism. Experiments on the NADP-inhibition of the interaction of NADPH and cytochrome c with liver reductase indicate that NADP is competitive with NADPH and noncompetitive with cytochrome c. This result is consistent with the postulate of a sequential reaction for NADPH-cytochrome P-450 reductases of liver and tumors. These data support the conclusions that an active drug metabolism system is present in liver tumors and that the tumor systems are constituted like the liver system.
Mol Cell Biochem 1978 Dec 22
PMID:The drug metabolism systems of liver and liver tumors: a comparison of activities and characteristics. 74 99

The activities of isocitrate dehydrogenase (NAD), isocitrate dehydrogenase (NADP) and oxoglutarate dehydrogenase have been investigated in Saccharomyces cerevisiae grown in a variety of aerobic and hypoxic conditions, the latter including oxygen deprivation, high glucose concentration, addition of inhibitors of mitochondrial protein synthesis, respiratory inhibition by azide, and impaired respiration mutants. All hypoxic conditions led to a marked decrease of oxoglutarate dehydrogenase and significant decreases of the two isocitrate dehydrogenases. According to its kinetic properties, the NAD-isocitrate dehydrogenase will not be operative in hypoxia "in vivo". From these and other related facts it is concluded that hypoxic conditions in yeast generally lead to a splitting of the tricarboxylic acid cycle and that glutamate synthesis in these conditions takes place through the coupling of the NADP-linked isocitrate and glutamate dehydrogenases.
Mol Cell Biochem 1975 Feb 28
PMID:Isocitrate dehydrogenases and oxoglutarate dehydrogenase activities of baker's yeast grown in a variety of hypoxic conditions. 109 51

Mutants, designated tamAr, have been isolated on the basis of simultaneous resistance to toxic analogues thiourea, aspartate hydroxamate and chlorate with L-alanine as the sole nitrogen source. tamAr mutants are also resistant to methylammonium. This resistance of tamAr mutants is correlated with partially repressed activity of a number of enzyme and transport systems regulated by ammonium. Furthermore, tam-Ar mutants have low NADP-glutamate dehydrogenase (NADP-GDH) activity and also efflux ammonium under certain growth conditions. Mutants at the areA locus (areAr) have also been isolated on the basis of resistance to these analogues, with nitrate or L-aspartate as the nitrogen source. These, similar to tamAr lesions, result in resistance to methylammonium and are partially repressed for ammonium repressible system, but in contrast to tamAr, areAr alleles have wild-type NADP-GDH activity and normal ammonium efflux. tamAr and areAr mutants grow as wild type on all nitrogen or carbon sources tested, are recessive, and appear to be epistatic to all other mutations (gdhA1, meaA8 and meaB6) which result in derepressed levels of ammonium regulated system. Whereas tamAr and areAr phenotypes are additive, tamAr is epistatic to areAd phenotype.
Mol Gen Genet 1975 Sep 29
PMID:Studies of partially repressed mutants at the tamA and areA loci in Aspergillus nidulans. 110 54

A putative isocitrate dehydrogenase (IDH) cDNA from alfalfa has been cloned and sequenced. The derived amino acid sequence of 433 residues contains the isocitrate and isopropylmalate dehydrogenase signatures, is 63% identical to yeast mitochondrial NADP-IDH and shares high sequence identity with peptides of pig heart NADP-IDH. The sequence contains a potential N-terminal leader with similarities to a thylakoid transit peptide. IDH transcripts and NADP-IDH activity were detected in all alfalfa tissues examined, their levels depending upon the tissue type and its developmental stage. Transcripts and enzymatic activity were not induced on exposure of cell suspension cultures to a fungal elicitor. IDH is encoded by a small gene family in alfalfa.
Plant Mol Biol 1992 Dec
PMID:Molecular characterization and expression of an isocitrate dehydrogenase from alfalfa (Medicago sativa L). 128 35

The high prevalence of glucose 6-phosphate dehydrogenase (G6PD) deficiency in African populations is due almost entirely to the enzyme variant A-, which differs from the wild-type G6PD B by two amino acid replacements, 68 Val-->Met and 126 Asn-->Asp. The non-deficient polymorphic variant G6PD A contains only the mutation 126 Asn-->Asp. The frequencies of the G6PD A and of the G6PD A- genes in parts of Africa are both about 0.2. The 68 Val-->Met mutation has not been found in a B background. This could be because the 68 Val-->Met mutation happened to arise in an A gene in the first instance, or because the 68 Val-->Met mutation alone is not sufficient to cause G6PD deficiency. We have approached this question by producing G6PD B, A, A-, and G6PD 68 Val-->Met in a bacterial expression system and analysing their biochemical properties. With each single mutation we found a slight decrease in both the specific activity and the yield of enzyme when compared to G6PD B. When both mutations were introduced together, there was a roughly additive effect on specific activity, but a much more drastic effect on enzyme yield (4% of normal). This synergistic effect was also demonstrated on thermal stability, especially at low NADP concentrations. Comparable results were produced when the replacement 119 Gln-->Glu was studied instead of 126 Asn-->Asp. We infer that the coexistence of the two mutations is responsible for enzyme deficiency in G6PD A- because they act synergistically in causing instability of the enzyme.
Hum Mol Genet 1992 Jun
PMID:Both mutations in G6PD A- are necessary to produce the G6PD deficient phenotype. 130 73

The activity of pure calf-liver and Escherichia coli thioredoxin reductases decreased drastically in the presence of NADPH or NADH, while NADP+, NAD+ and oxidized E. coli thioredoxin activated both enzymes significantly, particularly the bacterial one. The loss of activity under reducing conditions was time-dependent, thus suggesting an inactivation process: in the presence of 0.24 mM NADPH the half-lives for the E. coli and calf-liver enzymes were 13.5 and 2 min, respectively. Oxidized E. coli thioredoxin fully protected both enzymes from inactivation, and also promoted their complete reactivation after only 30 min incubation at 30 degrees C. Lower but significant protection and reactivation was also observed with NADP+ and NAD+. EDTA protected thioredoxin reductase from NADPH inactivation to a great degree, thus indicating the participation of metals in the process; EGTA did not protect the enzyme from redox inactivation. Thioredoxin reductase was extensively inactivated by NADPH under aerobic and anaerobic conditions, thus excluding the participation of O2 or oxygen active species in redox inactivation. The loss of thioredoxin reductase activity promoted by NADPH was much faster and complete in the presence of NAD+ glycohydrolase, thus suggesting that inactivation was related to full reduction of the redox-active disulfide. Those results indicate that thioredoxin reductase activity can be modulated in bacteria and mammals by the redox status of NADP(H) and thioredoxin pools, in a similar way to glutathione reductase. This would considerably expand the regulatory potential of the thioredoxin-thioredoxin reductase system with the enzyme being self-regulated by its own substrate, a regulatory protein.
Mol Cell Biochem 1992 Jan 15
PMID:NADPH and oxidized thioredoxin mediate redox interconversion of calf-liver and Escherichia coli thioredoxin reductase. 131 49

A Saccharomyces cerevisiae glutamate auxotroph, lacking NADP-glutamate dehydrogenase (NADP-GDH) and glutamate synthase (GOGAT) activities, was complemented with a yeast genomic library. Clones were obtained which still lacked NADP-GDH but showed GOGAT activity. Northern analysis revealed that the DNA fragment present in the complementing plasmids coded for a 1.5kb mRNA. Since the only GOGAT enzyme so far purified from S. cerevisiae is made up of a small and a large subunit, the size of the mRNA suggested that the cloned DNA fragment could code for the GOGAT small subunit. Plasmids were purified and used to transform Escherichia coli glutamate auxotrophs. Transformants were only recovered when the recipient strain was an E. coli GDH-less mutant lacking the small GOGAT subunit. These data show that we have cloned the structural gene coding for the yeast small subunit (GUS2). Evidence is also presented indicating that the GOGAT enzyme which is synthesized in the E. coli transformants is a hybrid comprising the large E. coli subunit and the small S. cerevisiae subunit.
Mol Microbiol 1992 Feb
PMID:Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs. 134 1

The gene encoding the streptococcal flavoprotein NADH oxidase (NOXase), which catalyzes the four-electron reduction of O2-->2H2O, has been cloned and sequenced from the genome of Streptococcus (Enterococcus) faecalis 10C1 (ATCC 11700). The deduced NOXase protein sequence corresponds to a molecular mass of 48.9 kDa and contains three previously sequenced cysteinyl peptides obtained with the purified enzyme. In Escherichia coli, the expressed nox gene produced a catalytically active product, which retained its immunoreactivity to affinity-purified NOXase antisera. Alignment of the NOXase protein sequence with that of streptococcal NADH peroxidase (NPXase) revealed that the proteins are 44% identical. Among the most highly conserved segments is a sequence containing Cys42; this residue is known to exist as a stabilized cysteine-sulfenic acid (Cys-SOH) in NPXase and serves as the non-flavin redox center. In addition, three previously identified NPXase segments, known to be involved in FAD and NAD(P)-binding in other pyridine nucleotide-linked flavoprotein oxidoreductases, are strongly conserved in NOXase. Overall, the extensive homology observed between NOXase and NPXase suggests that the monomer chain fold of the oxidase closely resembles that of the peroxidase. Both sequences share limited but significant homology to those of glutathione reductase and other members of the flavoprotein disulfide reductase family. These and other considerations suggest that these two unusual streptococcal flavoproteins constitute a distinct class of FAD-dependent oxidoreductases, the flavoprotein peroxide reductases, easily contrasted with enzymes such as glutathione reductase and thioredoxin reductase.
J Mol Biol 1992 Oct 05
PMID:Molecular cloning and analysis of the gene encoding the NADH oxidase from Streptococcus faecalis 10C1. Comparison with NADH peroxidase and the flavoprotein disulfide reductases. 140 82


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