Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
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In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD+-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%-70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.
Mol Gen Genet 1986 Jul
PMID:A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae. 352 55

Escherichia coli K-12 converts L-fucose to dihydroxyacetone phosphate (C-1 to C-3) and L-lactaldehyde (C-4 to C-6) by a pathway specified by the fuc regulon. Aerobically, L-lactaldehyde serves as a carbon and energy source by the action of an aldehyde dehydrogenase of broad specificity; the product, L-lactate, is then converted to pyruvate. Anaerobically, L-lactaldehyde serves as an electron acceptor to regenerate NAD from NADH by the action of an oxidoreductase; the reduced product, L-12-propanediol, is excreted. A strain selected for growth on L-galactose (a structural analog of L-fucose) acquired a broadened inducer specificity because of an altered fucR gene encoding the activator protein for the fuc regulon (Y. Zhu and E. C. C. Lin, J. Mol. Evol. 23:259-266, 1986). In this study, a second mutation that abolished aldehyde dehydrogenase activity was discovered. The L-fucose pathway converts L-galactose to dihydroxyacetone phosphate and L-glyceraldehyde. Aldehyde dehydrogenase then converts L-glyceraldehyde to L-glycerate, which is toxic. Loss of the dehydrogenase averts the toxicity during growth on L-galactose, but reduces by one-half the aerobic growth yield on L-fucose. When mutant cells induced in the L-fucose system were incubated with radioactive L-fucose, accumulation of radioactivity occurred if the substrate was labeled at C-1 but not if it was labeled C-6. Complete aerobic utilization of carbons 4 through 6 of L-fucose depends not only on an adequate activity of aldehyde dehydrogenase to trap L-lactaldehyde as its anionic acid but also on the lack of L-1,2-propanediol oxidoreductase activity, which converts L-lactaldehyde to a readily excreted alcohol.
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PMID:Loss of aldehyde dehydrogenase in an Escherichia coli mutant selected for growth on the rare sugar L-galactose. 354 71

Dihydrofolate reductase (DHFR) (5,6,7,8-tetrahydrofolate: NADPH+-oxidoreductase; EC 1.5.1.3) was partially purified by affinity chromatography from three clones of the human malaria parasite Plasmodium falciparum. The three clones were representative of pyrimethamine-sensitive (clone 3D7) and pyrimethamine-resistant (clone HB3 and clone 7G8) parasites with ID50 values of 0.53 nM (3D7), 210 nM (HB3), and 540 nM (7G8), when tested in vitro against the drug. The specific activities of the partially purified DHFR differed by less than a factor of 2 between the sensitive clone 3D7 (442 +/- 39 nmol min-1 mg-1 protein) and the resistant clones HB3 (634 +/- 25 nmol min-1 mg-1 protein) and 7G8 (565 +/- 85 nmol min-1 mg-1 protein). The number of catalytic sites in partially purified DHFR from the three clones was similar and ranged from 151 to 194 pmol mg-1 protein. The Km value for NADPH was similar in all three clones (4.5-11.6 microM). The Km value for dihydrofolate was altered 13-fold comparing the sensitive clone 3D7 (3.2 +/- 0.6 microM) with the resistant clone HB3 (42.6 +/- 1.6 microM), with the Km for the resistant clone 7G8 falling in between (11.9 +/- 1.2 microM). The inhibition constants for pyrimethamine increased from 0.19 +/- 0.08 nM (3D7) to 2.0 +/- 0.3 nM (HB3) to 8.9 +/- 0.8 nM (7G8). The inhibition by pyrimethamine of the sensitive clone 3D7 was noncompetitive and competitive for the two other clones. The titration of partially purified DHFR with pyrimethamine revealed a 500-fold increase in the concentration of the drug needed to inhibit the DHFR activity by 50%, when the sensitive clone 3D7 (0.18 +/- 0.02 nM) was compared to the resistant clone 7G8 (95 +/- 16 nM). From the comparison of the specific activities and the catalytic center activities with the Km values for the substrate and the inhibition constants for pyrimethamine, both of which are altered in the resistant clones, we conclude that the molecular mechanism for pyrimethamine resistance in the three clones studied is not based on an overproduction of the DHFR but is due to a decreased affinity to antifolates by a structurally altered enzyme.
Mol Pharmacol 1987 Apr
PMID:Kinetic and molecular properties of the dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant clones of the human malaria parasite Plasmodium falciparum. 355 92

The mechanism by which the nephrotoxic S-conjugates S-(1,2-dichlorovinyl)-L-cysteine (DCVC) and S-(1,2-dichlorovinyl)-L-homocysteine (DCVHC) produce toxicity in rat kidney mitochondria was studied by examining their effects on mitochondrial function, structural integrity, and metabolism. Both S-conjugates inhibited succinate-linked state 3 respiration and impaired the ability of mitochondria to retain Ca2+ and to generate a membrane potential; 30-60 min were required for maximal expression of these functional changes. Mitochondrial structure was damaged, as indicated by enhanced polyethylene glycol-induced shrinkage of matrix volume and by leakage of protein and malic dehydrogenase from the matrix; 60-120 min were required for maximal expression of these structural changes. Much shorter incubation times (15-30 min) were required for DCVC and DCVHC to decrease ATP concentrations, to alter the concentrations of several citric acid cycle intermediates, and to inhibit succinate:cytochrome c oxidoreductase and isocitrate dehydrogenase activities. Lipid peroxidation and oxidation of glutathione to glutathione disulfide also occurred. The relative time courses of these pathological changes indicate that the initial effects of DCVC and DCVHC in renal mitochondria are the inhibition of energy metabolism and the oxidation of glutathione. These changes then lead to alterations in mitochondrial function and ultimately to irreversible damage to mitochondrial structure.
Mol Pharmacol 1987 Oct
PMID:Mechanism of S-(1,2-dichlorovinyl)-L-cysteine- and S-(1,2-dichlorovinyl)-L-homocysteine-induced renal mitochondrial toxicity. 367 Feb 84

Several fragments of the human dihydrofolate reductase gene (tetrahydrofolate dehydrogenase, 5,6,7,8-tetrahydrofolate NADP+ oxidoreductase, EC 1.5.1.3) were isolated from gene-amplified KB7B cells and characterized. Recombinant plasmids containing intron sequences were constructed. Probes prepared from these plasmids were tested for dihydrofolate reductase precursor mRNA specificity via solution hybridization studies and Northern blot analysis. One probe, p0.69EH, was shown to be specific for dihydrofolate reductase RNA by its greatly enhanced level of hybridization with total RNA from dihydrofolate reductase gene-amplified versus non-amplified cells. In addition, solution hybridization studies with various classes of RNA and Northern blot analysis revealed that p0.69EH hybridizes predominantly with polyadenylated, high molecular weight, nuclear RNA species. Subsequent solution hybridization studies revealed a disproportionate 5-fluorouracil-induced increase in dihydrofolate reductase intron-containing RNA over dihydrofolate reductase mRNA. These results suggest that 5-fluorouracil incorporation into RNA may inhibit the conversion of precursor mRNA to mature mRNA.
Mol Pharmacol 1986 Jun
PMID:5-Fluorouracil augmentation of dihydrofolate reductase gene transcripts containing intervening sequences in methotrexate-resistant KB cells. 371 5

We show that the L-(+)-lactate dehydrogenase (EC 1.1.1.27;L-lactate: NAD+-oxidoreductase) of Plasmodium falciparum (LDH-P) is encoded in the parasite genome. A monoclonal antibody (McAb 7.2) has been shown to bind the LDH-P subunit which has an apparent molecular mass of 35 kDa. A polyclonal antiserum raised against affinity purified LDH-P has been used to isolate cDNA clones containing LDH-P epitopes from a lambda gt11Tn5 expression library. DNA sequence analysis of one clone, lambda LDH-P.1, reveals a single open reading frame which shows a degree of homology to the N-terminal domain of known LDH amino acid sequences.
Mol Biochem Parasitol 1985 May
PMID:Cloning studies on the gene coding for L-(+)-lactate dehydrogenase of Plasmodium falciparum. 389 92

22 revertants of Saccharomyces cerevisiae with intragenic suppressors (supa) of cob exon mutations (G. Burger, Mol. Gen. Genet., in the press) were analyzed. They display either a reduced amount of cytochrome b, or a shifted maximum absorption wavelength of total cytochrome b or a reduced growth rate on glycerol. The relationship of physico-chemical properties (content, light absorption and midpoint potential of cytochromes bK and bT) and functional properties (electron transport and energy yield) has been examined. In seven of eight revertants with a shifted maximum absorption wavelength of cytochrome b neither growth rate nor electron transfer activity was affected. In 13 of 14 revertants, reduced content of cytochrome b corresponds to a reduced electron transport rate through the cytochrome bc1 segment. A lower enzymatic activity, which is not due to a quantitative but to a qualitative alteration of cytochrome b was found in two revertants. Two revertants show electron transport rates of wild-type level concomitant with a reduced growth rate on glycerol, obviously due to a less efficient energy coupling. All revertants were shown to contain a high and a low potential cytochrome b, referred to as bK and bT. Those cob-/supa mutations which shift the maximum absorption wavelength or diminish the content of cytochrome b affect both b cytochromes in all cases. The results support that electron transport and energy conservation are catalyzed by the unity of cytochrome bK and bT and that both heme centers are bound to an identical apoenzyme. Comparing electron flow rates of succinate:cytochrome c oxidoreductase and NADH:cytochrome c oxidoreductase in cob- mutants and two revertants provides evidence that ubiquinone does not constitute a homogeneous pool, suggested by the dissimilar interaction of both dehydrogenases with the bc1 segment.
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PMID:Cytochrome b of cob revertants in yeast. Bioenergetic characterization of revertants with reduced content and shifted maximum absorption wavelength of cytochrome b. 608 48

Cells of the aerotolerant anaerobe Giardia lamblia respire in the presence of oxygen. Endogenous respiration is stimulated by glucose but not by other carbohydrates and Krebs cycle intermediates. Endogenous and glucose-stimulated respiration are insensitive to cyanide, malonate, and 2,4-dinitrophenol, but are inhibited by atabrin and iodoacetamide. G. lamblia produces ethanol, acetate and CO2 both aerobically and anaerobically either from endogenous reserves or exogenous glucose. Molecular hydrogen is not produced. The following enzyme activities were detected in homogenates: hexokinase, fructose-biphosphate aldolase, pyruvate kinase, phosphoenolpyruvate carboxykinase, malate dehydrogenase, malate dehydrogenase (decarboxylating), pyruvate synthase, acetyl-CoA synthetase, alcohol dehydrogenase (NADP+), NADH dehydrogenase, NADPH dehydrogenase, NADPH oxidoreductase and superoxide dismutase. The enzymes of energy and carbohydrate metabolism are nonsedimentable (109 000 x g for 30 min). Activities of lactate dehydrogenase, hydrogenase, phosphate acetyltransferase, acetate kinase, citrate synthase, succinate dehydrogenase, fumarate hydratase and catalase were below the limits of detection. The results suggest the occurrence of glycolysis, energy production by substrate level phosphorylation and a flavin, iron-sulfur protein mediated electron transport system as well as the absence of cytochrome mediated oxidative phosphorylation and functional Krebs cycle.
Mol Biochem Parasitol 1980 Mar
PMID:Energy metabolism of the anaerobic protozoon Giardia lamblia. 610 7

After a single intravenous injection of suramin the rate of removal of the drug from the plasma into other tissue compartments of the rat is independent of initial concentration. The data can be fitted to the sum of two exponential functions, consistent with a two-compartment, open model system. Trypanosomes take up only small amounts of suramin in vivo and do not actively concentrate the drug within the cell. Uptake is apparently by a non-saturable process that decreases with time and is dependent on the amount of suramin already taken up. Once within the cell, suramin progressively inhibits respiration and glycolysis, such that, for a given exposure in vivo, inhibition of oxygen consumption is proportional to the total amount of suramin absorbed. It can be calculated that only a fraction (4--9%) of this total is required to inhibit respiration to the extent found in broken cell preparations. The combined inhibition of two key enzymes in glycolysis--the sn-glycerol-3-phosphate oxidase (EC unassigned) and the glycerol-3-phosphate dehydrogenase (NAD+) (sn-glycerol-3-phosphate: NAD+ 2-oxidoreductase, EC 1.1.1.8)--are sufficient to account for the differential inhibition of glucose and oxygen consumption and of pyruvate production, together with the small, but significant, production of glycerol. Even at the highest dose of suramin tolerated by the rat, trypanosomes continue to increase exponentially in the bloodstream for at least 6 h. The mean doubling time is increased from 4.6 h to a maximum of about 12.5 h in rats treated with doses of suramin in the range 25--150 mg/kg. In the light of these and other findings, it is concluded that part of the trypanocidal action of suramin results from the inhibition of ATP production by glycolysis.
Mol Biochem Parasitol 1980 Oct
PMID:Uptake of the trypanocidal drug suramin by bloodstream forms of Trypanosoma brucei and its effect on respiration and growth rate in vivo. 610 10

The steady-state kinetics of tyrosine hydroxylase [L-tyrosine, tetrahydropteridine: oxygen oxidoreductase (3-hydroxylating); EC 1.14.16.2] frequently exhibits complex features which confound interpretation of the results. Using an assay-enzyme system which is essentially devoid of the major mitigating kinetic features, a comprehensive kinetics data base has been compiled. The studies employed L-tyrosine, 5,6,7,8-tetrahydrobiopterin, and oxygen as substrates, and 3-(3',4'-dihydroxyphenyl)L-alanine, a deazapterin, 3-iodo-L-tyrosine, and dopamine as product, substrate analogue, and product analogue inhibitors, respectively. All three reactants were varied pairwise, and all inhibitors (except dopamine) were tested with each of the three substrates as variable substrate. The entire data base was interpreted exclusively in terms of models for classic saturation kinetics of enzyme catalysis, providing an internally consistent kinetic model and evidence for a sequential mechanism with partially ordered sequences for substrate addition and product release. Some possible mechanisms and experimental variables relating these results to more complex kinetics of tyrosine hydroxylase are considered briefly.
Mol Pharmacol 1983 Jan
PMID:Steady-state kinetics of bovine striatal tyrosine hydroxylase. 613 41


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