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)

The intracellular synthesis and retention of polygammaglutamyl derivatives of methotrexate and their interactions with H2 folate reductase was evaluated in the Ehrlich ascites tumor cell and the isolated rat hepatocyte. Methotrexate polyglutamates were detected within 15 minutes in hepatocytes exposed to 1 microM methotrexate, and continued to accumulate for at least 60 minutes producing a large transmembrane gradient. These derivatives appeared to be preferentially retained within the cell even under conditions where release of intracellular methotrexate was induced by dibutyryl cyclic AMP or isobutyl methyl xanthine. Deoxycholate and bromosulfophthalein, compounds which inhibited methotrexate influx into hepatocytes, reduced the ratio of methotrexate polyglutamates to methotrexate, suggesting that these agents also inhibit the metabolism of methotrexate. In studies with the Ehrlich ascites tumor accumulation of methotrexate polyglutamates was increased over 5-fold by the addition of 5 mM L-glutamine or L-glutamate and exhibited a positive correlation with the extracellular concentration of methotrexate. Vincristine and probenecid, agents that increase the intracellular levels of methotrexate by inhibiting efflux, also produced a marked increase in methotrexate polyglutamates. When Ehrlich ascites tumor cells were exposed to 10 microM methotrexate and 5 mM L-glutamine intracellular polyglutamates were detected within 10 minutes and their levels increased linearly over 4 hours. As these derivatives accumulated, there was a decline in intracellular methotrexate due at least in part to a replacement of methotrexate on H2 folate reductase by polyglutamates and subsequent efflux of the previously bound methotrexate from the cell. When ppolyglutamate derivatives were in excess of the H2 folate reductase binding capacity and extracellular methotrexate removed, methotrexate rapidly exited the cell whereas the majority of its metabolites were retained and eventually saturated the major portion of the enzyme. These studies indicate that (1) intracellular methotrexate is rapidly converted to polygammaglutamyl derivatives, (2) these metabolites effectively compete with methotrexate for binding sites on H2 folate reductase, (3) these derivatives are retained within the cell more effectively than methotrexate, and (4) vincristine and probenecid may be potentially useful for selectively increasing methotrexate polyglutamates in tumor cells.
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PMID:Characteristics of the accumulation of methotrexate polyglutamate derivatives in Ehrlich ascites tumor cells and isolated rat hepatocytes. 619 86

Nifurtimox and nitrofurantoin are reduced by intact rat liver mitochondria to nitro anion radicals whose autoxidation generates superoxide anion as detected by direct electron spin resonance spectroscopy and by spin-trapping experiments, respectively. Although nitroreduction occurred in the presence of respiratory substrates such as beta-hydroxybutyrate, malate-glutamate, succinate, or endogenous substrates, nitro anion radical formation activity was much greater on addition of exogenous reduced pyridine nucleotides. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H nitroreductase reactions unless the mitochondria were solubilized by detergent. In addition, NAD(P)H nitroreductase activity was detected in the crude mitochondrial outer membrane fraction, with a higher activity than in mitoplasts and intact mitochondria. These results provide direct evidence of a nitrofuran reductase activity associated with the mitochondrial outer membrane that is far more important than that of respiratory chain enzymes.
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PMID:Reduction of nifurtimox and nitrofurantoin to free radical metabolites by rat liver mitochondria. Evidence of an outer membrane-located nitroreductase. 632 75

Fluorimetric titration has been used to measure the dissociation constants for the complexes of folate, pteroyltriglutamate and pteroylheptaglutamate with dihydrofolate reductase purified from Lactobacillus casei, Streptococcus faecium (isoenzyme 2) and bovine liver. Effects of pH, temperature, salt concentration and second ligands have been examined. The method is shown to be unsuitable for methotrexate complexes. The polyglutamates do not bind more tightly than folate to the S. faecium reductase under any conditions examined, but bind somewhat more tightly than folate to the L. casei reductase at low pH (less than 7) and to the bovine liver enzyme at pH 7-9. Increasing concentrations of KC1 decrease the binding of all three ligands to the L. casei and bovine liver enzymes. Increasing pH markedly raises the dissociation constants for all complexes of the L. casei reductase, but has only slight effects on the complexes of the S. faecium reductase. Complexes of the bovine enzyme are affected to an intermediate degree by pH, but the folate complex is affected much more than those of the polyglutamates. Model building studies have been performed with a three-dimensional model of the complex of L. casei reductase with NADPH and methotrexate. Additional glutamyl groups were added in gamma-linkage to the glutamate moiety of the complexed methotrexate. A proposed mode of binding of the pteroyl polyglutamates is discussed and sequence comparisons are used to predict residues that might be involved in polyglutamate binding by reductase from other sources.
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PMID:Molecular basis for the interaction of polyglutamates of folic acid and its analogs with dihydrofolate reductase. 641 19

13C nuclear magnetic resonance (NMR) of methotrexate, trimethoprim, and pyrimethamine enriched 90% with 13C at C2 has provided a sensitive means of detecting the state of protonation of the heterocyclic rings of these inhibitors. In each case, protonation of N1 causes an upfield movement of the chemical shift of C2 by more than 6 ppm. By this method it has been shown that, at pH values up to 9.2, methotrexate is bound to bovine liver dihydrofolate reductase with N1 of the inhibitor protonated, just as in the case of the complex with reductase from Streptococcus faecium and Lactobacillus casei. Furthermore, trimethoprim bound to reductase from any of the three sources, and pyrimethamine bound to either of the bacterial reductases also have N1 protonated even at pH values up to 10. This implies that in all cases there is a strong interaction between protonated N1 of the inhibitor and the carboxylate group of the active site aspartate or glutamate. In every case pKa of the bound inhibitor is increased by several units, a finding in accord with crystallographic evidence that inhibitor bound to L. casei reductase is in a hydrophobic environment and that N1 is not hydrogen-bonded to water. It was confirmed by titration of protein fluorescence that trimethoprim has greater affinity for bacterial reductase than for vertebrate (bovine) reductase, and that this selectivity is more marked in ternary complexes in which NADPH is also bound to the active site. However, the data cited above indicate that this difference in affinities is not due to a weaker ionic interaction between protonated N1 of trimethoprim and the bovine enzyme. Instead, binding of the trimethoprim side chain to hydrophobic sites on the enzyme must provide less binding energy in the case of the mammalian enzyme.
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PMID:Protonated state of methotrexate, trimethoprim, and pyrimethamine bound to dihydrofolate reductase. 641 76

The synthesis of 11-thiohomoaminopterin (1), which is a close analogue of 11-thiohomofolic acid (2), has been carried out by modification of the Boon-Leigh procedure. Treatment of 1-chloro-4-[p-(carbomethoxy)thiopenoxy]-2-butanone (5) with sodium azide gave 1-azido-4-[p-(carbomethoxy)thiophenoxy]-2-butanone (6). After protection of the carbonyl group of 6, the product 7 was catalytically hydrogenated to 1-amino-4-[p-(carbomethoxy)thiophenoxy]-2-butanone ketal (3). Reaction of 32 with 6-chloro-2,4-diaminmo-5-nitropyrimidine gave the desired pyrimidine intermediate, which was elaborated to 4-amino-4-deoxy-11-thiohomopteroic acid (20) by standard procedures. Alternately, 1-azido-4-[p-(carbomethoxy)thiophenoxy]-2-butanone ketal (7) was hydrolyzed to the corresponding acid (8) and coupled with diethyl L-glutamate to obtain diethyl N-[p-(1-azido-2-oxo-4-thiobutanoyl)benzoyl]-L-glutamate ketal (10), which was used for the large-scale preparation of 11-thiohomoaminopterin (1). Although 11-thiohomoaminopterin showed antifolate activity against two folate-requiring microorganisms and inhibited Lactobacillus casei dihydrogolate reductase, it did not exhibit any antitumor activity against L-1210 lymphoid leukemia in mice at a maximum dose of 48 mg/kg.
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PMID:Folate analogues altered in the C9-N10 bridge region. 16. Synthesis and antifolate activity of 11-thiohomoaminopterin. 677 88

The binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to dehydrofolate reductase from Lactobacillus casei MTX/R in the presence of a series of co-enzymes and coenzyme analogues has been measured fluorometrically. These two ligands, which can be regarded as "fragments" of the powerful inhibitor methotrexate, have been shown to bind cooperatively in the absence of coenzyme [Birdsall, B., Burgen, A. S. V., Rodrigues de Miranda, J., & Roberts, G. C. K. (1978) Biochemistry 17, 2102], p-amino-benzoyl-L-glutamate binding 58 times more tightly in the presence of 2,4-diaminopyrimidine than in its absence. In the presence of coenzymes, this cooperativity ranges from 1.8- to 428-fold. The effects of coenzymes on individual binding steps range from an 8-fold decrease in binding constant to a 23-fold increase. The structural specificity of these effects are discussed in terms of a model involving ligand-induced conformational changes and compared with the effects on trimethoprim and methotrexate binding described in the preceding paper [Birdsall, B., Burgen, A. S. V., & Roberts, G. C. K. (1980) Biochemistry (first paper of four in this issue)].
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PMID:Effects of coenzyme analogues on the binding of p-aminobenzoyl-L-glutamate and 2,4-diaminopyrimidine to Lactobacillus casei dihydrofolate reductase. 677 49

A proline dehydrogenase which catalyzes the nicotinamide adenine dinucleotide (NAD) dependent oxidation of proline and the NADH-dependent reduction of delta 1-pyrroline 5-carboxylic acid (PCA) was purified from extracts of Clostridium sporogenes. Following purification, only one protein band was found on analytical polyacrylamide disc gels and on sodium dodecyl sulfate (SDS) - polyacrylamide disc gels. Sucrose density gradient centrifugation and SDS-gel electrophoresis indicated that the enzyme has a molecular weight of approximately 217 000 and consists of two subunits of equal size. During purification of proline dehydrogenase on hydroxylapatite the ratio of dehydrogenase activity to reductase activity decreases significantly, and a similar change in ratio was brought about by storage of partially purified enzyme preparations in low ionic strength buffers. Subsequent purification did not change the ratio. The dehydrogenase activity of proline dehydrogenase was inhibited by L-glutamate (Ki = 0.32 mM at pH 7.4 and Ki = 0.65 mM at ph 10.2). However, the reductase activity of the purified enzyme was not affected by 100 mM L-glutamate.
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PMID:Purification and partial characterization of proline dehydrogenase from Clostridium sporogenes. 689 80

A new peroxide compound (ML-X) was isolated from an autoxidation product of methyl linoleate and was determined as methyl 9-hydroperoxy-12, 13-epoxy-10-octadecenoate. This compound inhibited state 3 respiration of rat heart- and liver mitochondria when glutamate and malate were used as substrates, but not when the substrate was succinate. State 4 respiration of mitochondria was not affected when glutamate-malate was used as the substrate, but it was stimulated when the substrate was succinate. ML-X inhibited oxidative phosphorylation of the mitochondria and abolished the membrane potential formed by respiration or by added ATP. NADH oxidase activity of submitochondrial particles was inhibited by ML-X but succinate oxidase activity was not inhibited. NADH-acceptor reductase activities of submitochondrial particles were inhibited by ML-X to the same extents as by rotenone. These findings show that ML-X has dual effects on mitochondrial respiration as (1) an inhibitor of NADH dehydrogenase complex and (2) an uncoupler. Neither methyl linoleate monohydroperoxide nor methyl epoxy stearate has such effects. ML-X is a new type of inhibitor-uncoupler of mitochondrial respiration in which hydroperoxy- and epoxy groups co-operate.
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PMID:Methyl hydroperoxy-epoxy-octadecenoate as an autoxidation product of methyl linoleate: a new inhibitor-uncoupler of mitochondrial respiration. 717 40

The possible effect of practolol (ICI 50,172) on mitochondrial metabolism was studied. The drug inhibited the oxidation of glutamate, alpha-ketoglutarate and succinate by heart mitochondria. The polarographic determinations showed that practolol is an inhibitor of the oxidative phosphorylation. The activity of NADH-oxidase, NADH-ferricyanide reductase, NADH-cytochrome c redutase was inhibit by the drug; no effect was observed on the succinate dehydrogenase. The electron microscopy of isolated mitochondria treated with practolol showed that the drug promote conformational changes of the mitochondrial membrane, probably concerning with the detergent characteristics of the drug.
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PMID:Studies of practolol on mitochondrial metabolism. 740 72

The biosynthesis of delta-aminolevulinate (ALA), via the C-5 pathway, requires tRNA(Glu) as a cofactor for the glutamyl tRNA(Glu) synthetase and the glutamyl tRNA(Glu) reductase which are the first two enzymes in this three step pathway. These two enzymes form a ternary complex with the tRNA(Glu) in Chlamydomonas reinhardtii suggesting that the recognition elements on the tRNA cofactor are different for each enzyme. Chemical modification and comparative studies with tRNA(Glu)s from a number of species were used to determine the nucleotides involved in the recognition of the barley chloroplast tRNA(Glu) by the barley enzymes. The barley chloroplast tRNA(Glu) is chemically modified both before and after ligation to glutamate with monobromobimane or CNBr. The chemically modified tRNA(Glu) is a poor substrate for the glutamyl-tRNA synthetase and the chemically modified glutamyl-tRNA(Glu) is used as a substrate for glutamyl-tRNA(Glu) reductase. The tRNA(Glu) from the chloroplasts if barley, Chlamydomonas reinhardtii, tobacco, cucumber, wheat and spinach and tRNA(Glu) from Synechocystis PCC6803, Escherichia coli, barley germ and bakers yeast and the barley chloroplast tRNA(Gln) are all effective substrates for the barley chloroplast glutamyl-tRNA synthetase. A comparison of the sequences of these tRNAs shows 19 conserved bases and five of these bases, G10, A26, U34, U35 and A37 are suggested as recognition elements of barley glutamyl tRNA(Glu) synthetase by assuming a similar binding orientation as in the crystal structure of the E. coli tRNA(Gln) GlnRS complex. The glutamyl-tRNA(Glu) from E. coli, bakers yeast and barley germ and the barley chloroplast glutamyl-tRNA(Gln) are not effective substrates for the barley chloroplast glutamyl-tRNA(Glu) reductase. A comparison of the sequences of these four tRNA species with the sequences of the tRNA(Glu) species that can be used as substrate by the glutamyl-tRNA(Glu) reductase yields seven common differences in the primary sequence. These 7 nucleotides, A7-U66, U29-A41, A53-U61, and U72 are expected to be required for recognition by the barley chloroplast glutamyl-tRNA(Glu) reductase.
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PMID:Nucleotides of tRNA (Glu) involved in recognition by barley chloroplast glutamyl-tRNA synthetase and glutamyl-tRNA reductase. 754 9


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