Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:Q8NEX9 (reductase)
 26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

gamma-Glutamyl phosphate reductase, the second enzyme of proline biosynthesis, catalyses the formation of l-glutamic acid 5-semialdehyde from gamma-glutamyl phosphate with NAD(P)H as cofactor. It was purified 150-fold from crude extracts of Pseudomonas aeruginosa PAO 1 by DEAE-cellulose chromatography and hydroxyapatite adsorption chromatography. The partially purified preparation, when assayed in the reverse of the biosynthetic direction, utilized l-1-pyrroline-5-carboxylic acid as substrate and reduced NAD(P)(+). The apparent K(m) values were: NAD(+), 0.36mm; NADP(+), 0.31mm; l-1-pyrroline-5-carboxylic acid, 4mm with NADP(+) and 8mm with NAD(+); P(i), 28mm. 3-(Phosphonoacetylamido)-l-alanine, a structural analogue of gamma-glutamyl phosphate, inhibited this enzyme competitively (K(i)=7mm). 1-Pyrroline-5-carboxylate reductase (EC 1.5.1.2), the third enzyme of proline biosynthesis, was purified 56-fold by (NH(4))(2)SO(4) fractionation, Sephadex G-150 gel filtration and DEAE-cellulose chromatography. It reduced l-1-pyrroline-5-carboxylate with NAD(P)H as a cofactor to l-proline. NADH (K(m)=0.05mm) was a better substrate than NADPH (K(m)=0.02mm). The apparent K(m) values for l-1-pyrroline-5-carboxylate were 0.12mm with NADPH and 0.09mm with NADH. The 3-acetylpyridine analogue of NAD(+) at 2mm caused 95% inhibition of the enzyme, which was also inhibited by thio-NAD(P)(+), heavy-metal ions and thiol-blocking reagents. In cells of strain PAO 1 grown on a proline-medium the activity of gamma-glutamyl kinase and gamma-glutamyl phosphate reductase was about 40% lower than in cells grown on a glutamate medium. No repressive effect of proline on 1-pyrroline-5-carboxylate reductase was observed.
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PMID:Biosynthesis of proline in Pseudomonas aeruginosa. Properties of gamma-glutamyl phosphate reductase and 1-pyrroline-5-carboxylate reductase. 11 73

The nucleotides DSI and DSII induced during a slowdown in growth of E. coli have been characterized using chemical and biochemical analysis and by enzymic and alkaline fragmentation. DSI consists a coenzyme A and glutathione joined by a disulfide linkage. DSI could be isolated either containing Fe(III) with an A250:260 ratio of 1.05 or not containing iron with an A250:260 of 0.87. DSII (isolated in 10% the yield of DSI) is a coenzyme A disulfide dimer that also contains two molecules of glutamic acid. DSI was a substrate for NADPH-dependent CoAS-SG reductase (EC 1.6.4.6) which was present in crude extracts of E. coli. The specific activity of CoAS-SG reductase increased during growth from early log phase into stationary phase and during a shift from aerobic to anaerobic growth.
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PMID:Identification of a coenzyme A--glutathione disulfide (DSI), a modified coenzyme A disulfide (DSII), and a NADPH-dependent coenzyme A--glutathione disulfide reductase in E. coli. 33 50

A competitive protein binding assay has been developed for methotrexate based on the tight binding of this drug to Lactobacillus casei dihydrofolate reductase (= tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase; EC 1.5.1.3). Free drug may be separated from that bound to reductase by adsorption with dextran--albumin coated charcoal. Scatchard plot analysis of the enzyme--drug interaction confirmed the presence of a single homogeneous class of binding sites with an association constant Ka of 2.1 X 10(8) M-1. This high affinity binding permits detection of methotrexate in the range of 0.3--30 pmol with a coefficient of variation of 15% or less. The predominant circulating folate, 5-methyl tetrahydrofolate, and the clinically useful rescue agent leucovorin (5-formyl tetrahydropteroyl-glutamic acid) do not interfere with the assay, nor does the methotrexate metabolite 4-amino-4-deoxy-10-methylpteroic acid. Assay of clinical samples, including plasma and cerebrospinal fluid, showed close agreement between the previously described enzyme inhibition assay and the more rapid competitive binding method.
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PMID:Competitive protein binding assay for methotrexate. 81 Aug 4

The Rhodospirillum rubrum structural gene puh, coding for the photoreaction center H polypeptide, and three other putative genes that surround puh were cloned and sequenced. The deduced 257 amino acid H polypeptide has a molecular weight of 27,909, in close agreement with polyacrylamide gel electrophoresis determination. Hydropathy plots predict a single hydrophobic alpha helix. The H polypeptide of Rhodospirillum rubrum shares only 23% of its residues with all three of the H polypeptides from Rhodopseudomonas viridis, Rhodobacter capsulatus, and Rhodobacter sphaeroides. Despite this apparent low degree of similarity, statistical analysis leaves no doubt about their close relatedness. Interspecies evolutionary distance, assessed by this analysis, confirms the closeness of the two Rhodobacter species, Rhodospirillum rubrum and Rhodopseudomonas viridis being approximately equidistant from them. Three regions of the H polypeptide are highly conserved in all four species. They correspond to known contact points of H with the complex of the other two (L and M) subunits on the cytoplasmic side of the membrane. A glutamic acid residue (H polypeptide residue 177), conserved in the other bacteria and suggested to be involved in the binding of secondary quinone QB, is replaced by serine in Rhodospirillum rubrum. The open reading frames G115, I2372, and I3087 are predicted to, respectively, encode polypeptides of 480, 224, and 155 residues coiled in 10, 2, and 1 transmembrane helices. Open reading frame G115 shares 56% identical residues with F1696, a sequence arranged in the genome of Rhodobacter capsulatus. The gene product of ORF I3087 is predicted to share highly similar sequences with nitrogenase reductase (encoded by nifH) of 11 different bacterial species and is suggested to have a regulatory function.
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PMID:The puh structural gene coding for the H subunit of the Rhodospirillum rubrum photoreaction center. 190 63

The reduction of Escherichia coli thioredoxin by thioredoxin reductase was studied by stopped-flow spectrophotometry. The reaction showed no dependence on thioredoxin concentration, indicating that complex formation was rapid and occurred during the dead time of the instrument. The kobs for the reaction of approximately 20 s-1 probably reflects the rate of electron transfer from thioredoxin reductase to thioredoxin and agrees with the kcat observed by steady-state kinetics. The reaction rate was unaffected by increasing the ionic strength, suggesting a lack of electrostatic stabilization in the interaction of the two proteins. A mutant thioredoxin in which a positively charged lysine in the active-site region was changed to a glutamic acid residue resulted in an electrostatic destabilization. Thioredoxin K36E was still a substrate for the reductase, but binding was impaired so that the rate could be measured by stopped-flow techniques as reflected by a dependence on protein concentration. Raising the ionic strength in this reaction served to shield the negative charge and increased the rate of binding to the reductase.
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PMID:Kinetics of electron transfer from thioredoxin reductase to thioredoxin. 199 79

An incubation medium was established for the microphotometric demonstration of glutamate dehydrogenase (Gldh) in cryostat sections of the rat hippocampus which served as an exemplary brain region. The final incubation medium consisted of 100 mM L-glutamic acid monosodium salt, 5 mM NAD, 10 mM sodium azide (NaN3), 5 mM ADP, 20 mM sodium chloride, 0.15 mM phenazine methosulfate (PMS), 5 mM nitroblue tetrazolium chloride and 22% polyvinyl alcohol (PVA) in 0.05 M Hepes buffer; the final pH was 7.5. The study showed that in the histochemical demonstration of Gldh the use of relatively high PVA concentrations were necessary to avoid diffusion artefacts because Gldh seems to be only loosely bound to the mitochondrial matrix. The use of NaN3 as a blocker of the respiratory chain was indispensible, because without NaN3 most reduction equivalents were lost through the respiratory chain. With PMS as an exogenous electron carrier, the demonstrable Gldh activities increased significantly indicating that, in the case of Gldh, the endogenous NADH tetrazolium reductase was not sufficiently effective. Furthermore, it was shown that Gldh was affected by many small molecules (e.g. activation by sodium ions, inhibition by magnesium and calcium ions) so that minor variations of the incubation conditions may cause major differences in demonstrable activities.
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PMID:Microphotometric determination of enzymes in brain sections. III. Glutamate dehydrogenase. 233 53

The 8 kDa protein of beef heart ubiquinol:cytochrome c reductase was detected by means of a new SDS-PAGE [(1985) FEBS Lett. 190, 89-94] system and was isolated by a series of chromatographic steps involving dissociation of the complex by salt treatment. The amino acid sequence was determined by solid-phase Edman degradation of both the N-terminal part of the whole protein and proteolytic cleavage fragments of the protein. The protein consists of 78 amino acid residues: its Mr was calculated to be 7998. Structure predictions have been made from average and sided hydropathy profiles. The suggested structure encompasses an alpha-helix and a beta-strand, the latter comprising a glutamic acid residue situated in a relatively hydrophobic neighbourhood. This residue may be responsible for the fact that the 8 kDa protein is the first subunit of the whole reductase (consisting of 11 subunits) to be labelled by DCCD when the reductase is in free form or inlaid in phospholipid vesicles.
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PMID:Isolation and amino acid sequence of the 8 kDa DCCD-binding protein of beef heart ubiquinol:cytochrome c reductase. 299 28

CO dehydrogenase, a key enzyme of the acetyl-CoA pathway of autotrophic growth, has been methylated using 14CH3I or 14CH3-corrinoid enzyme plus ferredoxin. Acetyl-CoA was synthesized from the resulting 14CH3-CO dehydrogenase, CO, and CoASH, with about 50% yield of the available 14C and without addition of other enzymes except CO dehydrogenase disulfide reductase. Even the reductase could be replaced by dithioerythritol. Amino acid analysis of the 14CH3-CO dehydrogenase showed two radioactive peaks, one of which migrated as S-methylcysteine but very close to the methyl ester of glutamic acid. By oxidation with H2O2, the radioactive component of this peak was identified as S-methylcysteine sulfone. Amino acid analysis of the 14CH3-CO dehydrogenase after synthesis of acetyl-CoA demonstrated that there was a large decrease in radioactivity of the peak containing the S-methyl-cysteine. The compound present in the second peak has not been identified; there was no decrease in its radioactivity. By nonreducing gel electrophoresis of the 14CH3-CO dehydrogenase, followed by autoradiography, it was shown that the beta subunit is the methyl acceptor. These results demonstrate that a cysteine of the beta subunit is the methyl acceptor and that CO dehydrogenase per se catalyzes the synthesis of acetyl-CoA.
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PMID:Acetyl-CoA pathway of autotrophic growth. Identification of the methyl-binding site of the CO dehydrogenase. 318 78

The structural genes (luxA-E) of the Vibrio harveyi luminescent system coding for the luciferase (alpha, beta) and fatty acid reductase (r, s, t) polypeptides can be expressed exclusively in Escherichia coli under the T7 phage promoter providing a convenient method for identifying and locating these genes. luxD which codes for the acyltransferase enzyme producing fatty acids for the luminescent reaction was located immediately above the luciferase genes (luxA, B) with the two other fatty acid reductase genes (luxC, E) flanking these genes, in the same order as found for the Vibrio fischeri luminescent system. By hybridization with luxC DNA probes, a set of mRNAs coding for this gene was detected; part of this set of mRNAs extended downstream and complemented the set of mRNAs previously detected for the other lux structural genes. The luxD gene from a mutant (M17) which requires tetradecanoic acid for light emission was cloned into the T7 system, and upon expression it could be demonstrated that the lack of activity was due to synthesis of a full-length nonfunctional protein and not to introduction of a stop codon. The nucleotide sequence of the luxD gene of the native and mutant strains was determined and shown to consist of an open reading frame of 915 bases preceded by a Shine-Dalgarno sequence with very high homology to the ribosome binding site found for luxA and B. The 3'-sequence was identical to a 669-base open reading frame upstream of the luxA gene reported earlier by Cohn et al. (Cohn, D.H., Mileham, A.J., Simon, M.I., Nealson, K.H., Rausch, S.K., Bonam, D., and Baldwin, T.O. (1985) J. Biol. Chem. 260, 6139-6146). The luxD mutant arose by a single point mutation of G to A resulting in a change of glycine to glutamic acid.
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PMID:Organization of the lux structural genes of Vibrio harveyi. Expression under the T7 bacteriophage promoter, mRNA analysis, and nucleotide sequence of the luxD gene. 341 63

In an attempt to bring some insight into how peptidoglycan synthesis is controlled in Escherichia coli, simple parameters, such as cell peptidoglycan content, the pool levels of its seven uridine nucleotide precursors, and the specific activities of five enzymes involved in their formation, were investigated under different growth conditions. When exponential-phase cells with generation times ranging from 25 to 190 min were examined, the peptidoglycan content apparently varied as the cell surface area changed, and no important variations in the pool levels of the nucleotide precursors or in the specific activities of the five enzymes considered were observed. The peptidoglycan of exponential-phase cells accounted for 0.7 to 0.8% of the dry cell weight, whereas that of stationary-phase cells accounted for 1.4 to 1.9%. Depending on the growth conditions, the number of peptidoglycan disaccharide peptide units per cell varied from 2.4 X 10(6) to 5.6 X 10(6). The levels of the nucleotide precursor pools as well as the specific activities of the D-glutamic acid- and D-alanyl-D-alanine-adding enzymes varied little with the growth phase. The specific activities of UDP-N-acetylglucosamine transferase, UDP-N-acetylglucosamine-enolpyruvate reductase, and the diaminopimelic acid-adding enzymes decreased by 20 to 50% at most in the late stationary phase. The results are discussed in terms of the possible importance for cell survival of the maintenance of a high capacity for peptidoglycan synthesis, whatever its rate under various growth conditions, and of a balance between the synthesis and breakdown of peptidoglycan during active growth.
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PMID:Effect of growth conditions on peptidoglycan content and cytoplasmic steps of its biosynthesis in Escherichia coli. 389 26


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