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)

Antibodies directed against cytochrome P-450Cm1 and the NADPH-cytochrome P-450 reductase were used to study the induction and intracellular localization of these components of the alkane monooxygenase system in the yeast Candida maltosa. Transition from glucose to n-hexadecane utilization resulted in an about 100-fold increase of the immunodetectable P-450 form whereas the reductase was only moderately induced by a factor of about 5. P-450 but not the reductase was further increased by oxygen limitation during cultivation on n-hexadecane. Using an immunogold technique on ultrathin cryosections, P-450 was found to be concentrated in the nuclear envelope during the early phase of the induction process. However, after maximal induction, the highest labeling was observed in membranes of the endoplasmic reticulum closely associated with the peroxisomes and the plasma membrane. Double-labeling experiments revealed that P-450 and its reductase were distributed in the same regions of the endoplasmic reticulum.
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PMID:Immunocytochemical localization of alkane-inducible cytochrome P-450 and its NADPH-dependent reductase in the yeast Candida maltosa. 151 3

Substrate has recently been shown to affect (a) the high spin content of cytochrome P450 (b) the rate of first electron transfer when LM2 (P450 2B4) and reductase were in a preformed complex, and (c) the rate of functional complex formation between NADPH-cytochrome P450 reductase and cytochrome P450 LM2. When comparing the effect of substrate on each of these parameters, the strongest correlation was demonstrated between the rate of first electron transfer through the preformed complex and the rate of functional complex formation (W.L. Backes and C.S. Eyer, 1989, J. Biol. Chem. 264, 6252-6259). The relationship among high spin content, reduction rate, and the rate of functional complex formation was examined using a number of different cytochrome P450 isozymes. The goal of this study was to determine if the previously established relationship between reduction rate and the rate of reductase-P450 complex formation was a feature only of LM2, or a general characteristic of the cytochrome P450 system. Substrate addition caused an increase in first electron transfer for each of the isozymes examined, with high spin content being increased with cytochromes P450 2B1 (PBRLM5) and P450 2B2 (PBRLM6). Substrate addition to cytochrome P450 2C6 (PBRLM4) resulted in a small decrease in high spin content. P450 2B1 and P450 2B2 showed a positive correlation between substrate-mediated stimulation of reduction and high spin content, whereas P450 2C6 showed a negative correlation between these variables. Substrate also increased the rate of reductase-P450 association for each of the isozymes examined. When compared to the degree of stimulation of reduction through a preformed complex, a strong positive correlation was obtained with each isozyme examined. These results demonstrate that the increase in both the rate of functional reductase-P450 complex formation and the rate of first electron transfer is not simply a property of LM2, but appears to be a general characteristic of many cytochrome P450 isozymes.
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PMID:Relationship between the rate of reductase-cytochrome P450 complex formation and the rate of first electron transfer. 153 61

Glycerol can be oxidized by rat liver microsomes to formaldehyde in a reaction that requires the production of reactive oxygen intermediates. Studies with inhibitors, antibodies, and reconstituted systems with purified cytochrome P4502E1 were carried out to evaluate whether P450 was required for glycerol oxidation. A purified system containing phospholipid, NADPH-cytochrome P450 reductase, P4502E1, and NADPH oxidized glycerol to formaldehyde. Formaldehyde production was dependent on NADPH, reductase, and P450, but not phospholipid. Formaldehyde production was inhibited by substrates and ligands for P4502E1, as well as by anti-pyrazole P4502E1 IgG. The oxidation of glycerol by the reconstituted system was sensitive to catalase, desferrioxamine, and EDTA but not to superoxide dismutase or mannitol, indicating a role for H2O2 plus non-heme iron, but not superoxide or hydroxyl radical in the overall glycerol oxidation pathway. The requirement for reactive oxygen intermediates for glycerol oxidation is in contrast to the oxidation of typical substrates for P450. In microsomes from pyrazole-treated, but not phenobarbital-treated rats, glycerol oxidation was inhibited by anti-pyrazole P450 IgG, anti-hamster ethanol-induced P450 IgG, and monoclonal antibody to ethanol-induced P450, although to a lesser extent than inhibition of dimethylnitrosamine oxidation. Anti-rabbit P4503a IgG did not inhibit glycerol oxidation at concentrations that inhibited oxidation of dimethylnitrosamine. Inhibition of glycerol oxidation by antibodies and by aminotriazole and miconazole was closely associated with inhibition of H2O2 production. These results indicate that P450 is required for glycerol oxidation to formaldehyde; however, glycerol is not a direct substrate for oxidation to formaldehyde by P450 but is a substrate for an oxidant derived from interaction of iron with H2O2 generated by cytochrome P450.
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PMID:Role of cytochrome P450 in the oxidation of glycerol by reconstituted systems and microsomes. 153 67

Cytochrome P450IA1 (purified from hepatic microsomes of beta-naphthoflavone-treated rats) has been covalently modified with the lysine-modifying reagent acetic anhydride. Different levels of lysine residue modification in cytochrome P450IA1 can be achieved by varying the concentration of acetic anhydride. Modification of lysine residues in P450IA1 greatly inhibits the interaction of P450IA1 with NADPH-cytochrome P450 reductase. Modification of 1.0 and 3.3 mol lysine residues per mole P450IA1 resulted in 30 and 95% decreases, respectively, in 7-ethoxycoumarin hydroxylation by a reconstituted P450IA1/reductase complex. However, modification of 3.3 mol lysine residues per mole P450IA1 decreased only cumene hydroperoxide-supported P450-dependent 7-ethoxycoumarin hydroxylation by 30%. Spectral and fluorescence studies showed no indication of global conformational change of P450IA1 even with up to 8.8 mol lysine residues modified per mole P450IA1. These data suggest that at least three lysine residues in P450IA1 may be involved in the interaction with reductase. Identification of lysine residues in P450IA1 possibly involved in this interaction was carried out by [14C]acetic anhydride modification, trypsin digestion, HPLC separation, and amino acid sequencing. The lysine residue candidates identified in this manner were K97, K271, K279, and K407.
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PMID:The role of cytochrome P450 lysine residues in the interaction between cytochrome P450IA1 and NADPH-cytochrome P450 reductase. 155 Mar 61

The role of cytochrome b5 in adrenal microsomal steroidogenesis was studied in guinea pig adrenal microsomes and also in the liposomal system containing purified cytochrome P-450s and NADPH-cytochrome P-450 reductase. Preincubation of the microsomes with anti-cytochrome b5 immunoglobulin decreased both 17 alpha- and 21-hydroxylase activity in the microsomes. In liposomes containing NADPH-cytochrome P-450 reductase and P-450C21 or P-450(17) alpha,lyase, addition of a small amount of cytochrome b5 stimulated the hydroxylase activity while a large amount of cytochrome b5 suppressed the hydroxylase activity. The effect of cytochrome b5 on the rates of the first electron transfer to P-450C21 in liposome membranes was determined from stopped flow measurements and that of the second electron transfer was estimated from the oxygenated difference spectra in the steady state. It was indicated that a small amount of cytochrome b5 activated the hydroxylase activity by supplying additional second electrons to oxygenated P-450C21 in the liposomes while a large amount of cytochrome b5 might suppress the activity through the interferences in the interaction between the reductase and P-450C21.
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PMID:The role of cytochrome b5 in adrenal microsomal steroidogenesis. 155 20

31P NMR spectroscopy has been utilized in conjunction with site-directed mutagenesis and phospholipid analysis to determine structural aspects of the prosthetic flavins, FAD and FMN, of NADPH-cytochrome P450 reductase. Comparisons are made among detergent-solubilized and protease (steapsin)-solubilized preparations of porcine liver reductases, showing unequivocally that the 31P NMR signals at approximately 0.0 ppm in the detergent-solubilized, hydrophobic form are attributable to phospholipids. By extraction and TLC analysis, the phospholipid contents of detergent-solubilized rat liver reductase, both tissue-purified and Escherichia coli-expressed, have been determined to reflect the membranes from which the enzyme was extracted. In addition, the cloned, wild-type NADPH-cytochrome P450 reductase exhibits an additional pair of signals downfield of the normal FAD pyrophosphate resonances reported by Otvos et al. [(1986) Biochemistry 25, 7220-7228], but these signals are not observed with tissue-purified or mutant enzyme preparations. The Tyr140----Asp140 mutant, which exhibits only 20% of wild-type activity, displays no gross changes in 31P NMR spectra. However, the Tyr178----Asp178 mutant, which has no catalytic activity and does not bind FMN, exhibits no FMN 31P NMR signal and a normal, but low intensity, pair of signals for FAD. The latter experiments, taking advantage of mutations in residues putatively on either side of the FMN isoalloxazine ring, suggest subtle to severe changes in the binding of the flavin prosthetic groups and, perhaps, cooperative interactions of flavin binding to NADPH-cytochrome P450 reductase.
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PMID:31P NMR spectroscopic studies on purified, native and cloned, expressed forms of NADPH-cytochrome P450 reductase. 156 69

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.
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PMID:Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity. 162 48

NADPH-cytochrome P450 reductase was purified to apparent homogeneity and cytochrome P450 partially purified from whole rat brain. Purified reductase from brain was identical to liver P450 reductase by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot techniques. Kinetic studies using cerebral P450 reductase reveal Km values in close agreement with those determined with enzyme purified from rat liver. Moreover, the brain P450 reductase was able to function successfully in a reconstituted microsomal system with partially purified brain cytochrome P450 and with purified hepatic P450c (P450IA1) as measured by 7-ethoxycoumarin and 7-ethoxyresorufin O-deethylation. Our results indicate that the reductase and P450 components may interact to form a competent drug metabolism system in brain tissue.
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PMID:Reconstitution of the brain mixed function oxidase system: purification of NADPH-cytochrome P450 reductase and partial purification of cytochrome P450 from whole rat brain. 162 29

Cytochrome b558, which is considered to be an essential component of the phagocytic superoxide (O2-)-generating system, was highly purified from porcine neutrophils. The isolated cytochrome was resolved into two polypeptides with molecular masses of 60-90 and 19 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. For enzymatic reduction of purified cytochrome b558, we utilized hepatic NADPH-cytochrome P450 reductase purified from rat liver microsomes. More than 80% of the cytochrome was reduced by incubation with the reductase and NADPH under the anaerobic condition, and was quickly reoxidized by the air. As indicated by measurement of oxygen consumption, the purified cytochrome catalytically reduced oxygen at a rate equal to approximately 30% of the activity of the phorbol myristate acetate-activated cells on the basis of cytochrome b558 content. Electron paramagnetic resonance study with a spin trapping agent 5, 5-dimethyl-1-pyrroline-1-oxide demonstrated that O2- is the exclusive primary product in the reduction of oxygen by the cytochrome. This gives direct evidence that cytochrome b558 functions as the terminal oxidizing enzyme in the O2- -generating system of neutrophils. This also establishes a new functional class of heme proteins that catalyzes one-electron reduction of molecular oxygen.
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PMID:Superoxide production by cytochrome b558 purified from neutrophils in a reconstituted system with an exogenous reductase. 164 21

The toxicity of paraquat is due to the oxygen-derived radicals formed by the reaction of oxygen with bipyridylium radical cations. Although paraquat is known to cause lung toxicity, the related bipyridylium compounds such as diquat and morfamquat do not affect the lung as seriously, but rather cause liver toxicity. Paraquat, diquat, morfamquat, and benzyl viologen are reduced by rat hepatocytes to their respective radical cations. An intracellular component of the signal was detected from diquat and benzyl viologen radical cations. These radical cations generated inside the cell can cross the plasma membrane. Generation of the diquat radical cation by hepatocytes is not affected by the inhibition of cytochrome P-450 by carbon monoxide or metyrapone, suggesting that this enzyme is probably not involved in the reduction of diquat as had been proposed previously. The reduction of paraquat is generally attributed to NADPH-cytochrome P-450 reductase, and presumably diquat is also reduced by this flavoprotein. Some transition metal chelates such as ferric diethylenetriaminepentaacetic acid delay the detection of the diquat radical cation. This may be due to the reduction of the ferric chelate by the diquat radical cation resulting in the formation of the ferrous chelate and the parent bipyridylium dication. When all the ferric chelate has been reduced to the ferrous chelate, then the bipyridylium radical can be detected. Alternatively, if the ferric chelate enters the cell, it can compete with the parent bipyridylium dication for the reductase, which would also lead to delayed detection.
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PMID:Reduction of paraquat and related bipyridylium compounds to free radical metabolites by rat hepatocytes. 165 43


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