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

In the present study the interaction of NADPH-cytochrome reductase with phospholipids was investigated using 31P-NMR, thin-layer chromatography combined with chemical analysis, fluorescence spectroscopy and kinetic studies with purified rat liver cytochrome P450 IIB1. 31P-NMR analysis demonstrates that the composition of the phospholipids that remain associated to NADPH-cytochrome reductase upon its purification is significantly different from the phospholipid composition of the microsomal membrane. Thin-layer chromatography followed by chemical analysis of the phospholipid composition demonstrates that the isolated NADPH-cytochrome reductase was enriched in L-alpha-1,2-diacyl-sn-glycero-3-phosphoserine (acyl2GroPSer) and L-alpha-1,2-diacyl-sn-glycero-3-phosphoinositol (acyl2GroPIns) compared to the microsomal membrane. The observed preference of NADPH-cytochrome reductase for acyl2GroPSer and acyl2GroPIns appeared not to be a result of the procedure for solubilisation and/or purification of the protein. The specific interaction of NADPH-cytochrome reductase with acyl2GroPSer and acyl2GroPIns was further investigated by comparison of the effect of acyl2GroPSer and acyl2GroPIns with that of acyl2GroPCho and acyl2GroPEtn on the 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3- phosphocholine-(DphPamGroPCho)-dependent quenching of the tryptophan fluorescence of purified NADPH-cytochrome reductase. The results demonstrate that the addition of acyl2GroPSer or acyl2GroPIns affects the DphPamGroPCho-dependent quenching of the tryptophan fluorescence in a manner significantly different from the addition of acyl2GroPCho or acyl2GroPEtn. The relatively larger DphPamGroPCho-induced quenching of the tryptophan fluorescence of NADPH-cytochrome reductase in the presence of acyl2GroPSer and acyl2GroPIns must result from a change in the conformation of NADPH-cytochrome reductase induced by the latter two lipids. Finally, the possible consequences of this special interaction of acyl2GroPSer and acyl2GroPIns with NADPH-cytochrome reductase on the kinetic characteristics of the cytochrome P450 system were studied using cytochrome-P450-IIB1-dependent O-dealkylation of pentoxyresorufin as the model reaction. These studies demonstrate that a 1:1 mixture of acyl2GroPCho and acyl2GroPSer results in a significantly higher apparent maximum rate (V) of O-dealkylation than a 1:1 mixture of acyl2GroPCho and acyl2ProPEtn or acyl2GroPCho alone. This increase in the apparent V can be ascribed to an acyl2GroPSer-dependent improvement of the interaction of NADPH-cytochrome reductase with cytochrome P450. This improvement of the interaction of the proteins cannot, however, be exclusively ascribed to the negative charge of acyl2GroPSer, since the other negatively charged phospholipid investigated, namely acyl2GroPIns, resulted in a significant decrease in the apparent V.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:A specific interaction between NADPH-cytochrome reductase and phosphatidylserine and phosphatidylinositol. 828 20

In the present study the effect of changing the fatty acyl moiety of phosphatidylcholine from dilauroyl to distearoyl on the kinetic parameters of O-dealkylation of alkoxyresorufins and ethoxycoumarin dependent on reconstituted cytochromes P-450 IA1 and IIB1 has been investigated. The results demonstrate that (a) the maximum rate of O-dealkylation (V) for both P-450 enzymes was about two times higher in the L-alpha-dilauroyl-sn-glycero-3-phosphocholine (Lau2GroPCho) system and (b) changes in the fatty acyl moiety of phosphatidylcholine (acyl2GroPCho) from dilauroyl to distearoyl affected the apparent Km for the substrate (Kms) of P-450 IA1 and IIB1 in a different way. In addition, (c) the kinetic parameters appeared to be dependent on the acyl2GroPCho/P-450 ratio and a change in this ratio affected the kinetic parameters of P-450 IA1 and IIB1 in a different manner. From these last two observations it was concluded that the mechanism by which phospholipids influence P-450-IIB1-dependent O-dealkylation of ethoxycoumarin is different from that by which they influence P-450-IIB1-dependent O-dealkylation of this substrate. Furthermore, the results of the present study demonstrate that the increase in the rate of O-dealkylation of ethoxycoumarin, reported in the literature for reconstituted systems in the presence of Lau2GroPCho, results from an effect of Lau2GroPCho on both the Kms and the V. In a number of additional experiments possible mechanisms underlying the observed differential effect of Lau2GroPCho and Ste2GroPCho on the Kms and V of P-450 IA1 and IIB1 were investigated. This was done by studying the effect of the two acyl2GroPCho species on the kinetic parameters of some of the different steps of the P-450 cycle, namely substrate binding, oxygen binding and the rate of electron transfer. The results demonstrate an influence of Lau2GroPCho and Ste2GroPCho on (a) substrate binding to cytochrome P-450, (b) the affinity of cytochromes P-450 for NADPH-cytochrome reductase and thus on (c) the electron flow through the reconstituted system. Based on the results from these experiments it was concluded that the increased V of P-450 IA1 and IIB1 in the presence of Lau2GroPCho compared to the systems with Ste2GroPCho was at least in part due to an increased affinity of both P-450 enzymes for NADPH-cytochrome reductase in the presence of Lau2GroPCho compared to Ste2GroPCho.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Kinetics of cytochromes P-450 IA1 and IIB1 in reconstituted systems with dilauroyl- and distearoyl-glycerophosphocholine. 834 4

The present study demonstrates the presence of multiple forms of cytochrome P450 (P450) in human brain obtained at autopsy, the purification of various isoforms to apparent homogeneity, and the monooxygenase activities in reconstituted systems. Sequential chromatography on octylamino-Sepharose 4B, DEAE-Sephacel, and DEAE-cellulose yielded four isoforms of P450 (A, B, C, and D) with specific contents of 11.0, 9.4, 12.5, and 8.3 nmol of P450/mg protein, respectively. While the forms A, B, and C were apparently homogeneous as examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis; the P450D was not homogeneous. The apparent molecular masses of the four forms of P450 were 60,200 Da (P450A), 60,900 Da (P450B), 60,200 Da (P450C), and 61,000 Da (P450D), respectively. NADPH cytochrome P450 reductase (reductase) was also partially purified from the brain microsomes. Immunoblot analysis of the four forms of human purified P450, using antisera to purified rat liver P450 (IIB1 + IIB2), rat liver P450 (1A1 + 1A2), phenobarbital-inducible rat brain P450, human liver P450 IIE1, P450 1A2, P450 IIC, and P450 IIIA4, indicated differential immunological cross-reactivity. The monooxygenase activities of the purified human brain P450s were demonstrated with various substrates (aminopyrine, morphine, aniline, 7-ethoxycoumarin, and nifedipine) as examined in reconstituted systems consisting of purified human brain P450, purified rat brain NADPH cytochrome P450 reductase, deoxycholate, phospholipid, and NADPH.
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PMID:Purification of multiple forms of cytochrome P450 from a human brain and reconstitution of catalytic activities. 846 Sep 38

Hepatic cytochrome P-450 activity has been shown to be affected by various dietary factors including vitamin E. However, reports of the effect of dietary vitamin E on cytochrome P-450 activity have been inconsistent. The aim of the present study was to investigate the influence of dietary vitamin E on rat hepatic cytochrome P-450 activity. Three groups of six male weanling Sprague-Dawley rats were fed semipurified diets containing 0, 100, or 1,500 ppm vitamin E for eight weeks. Vitamin E was given in the form of alpha-tocopheryl acetate. Dietary vitamin E significantly affected liver vitamin E content (p < 0.05) but had no effect on rat hepatic total P-450 content, N-nitrosodimethylamine demethylase, and NADPH-cytochrome-P-450 reductase activities. Hepatic pentoxyresorufin O-dealkylase and glutathione S-transferase activities were significantly greater in rats fed 100 and 1,500 ppm vitamin E than in rats fed no vitamin E (p < 0.05). Dietary vitamin E induced changes in hepatic phospholipid fatty acid composition. Hepatic phospholipid linoleate was significantly greater in rats fed 0 and 1,500 ppm vitamin E than in rats fed 100 ppm vitamin E (p < 0.05). Hepatic phospholipid eicosapentaenoate was increased significantly by dietary vitamin E (p < 0.05). Hepatic thiobarbituric acid-reactive substance was significantly greater in rats fed no vitamin E than in rats fed 100 and 1,500 ppm vitamin E (p < 0.05). The results suggest that vitamin E may influence cytochrome P-450 IIB1 enzyme activity and may affect hepatic phospholipid fatty acid composition.
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PMID:Effect of vitamin E on rat hepatic cytochrome P-450 activity. 979 69

While cancer drug resistance has been extensively studied in cell culture, little is known about more clinically relevant in vivo resistance. The in vivo resistance of a murine mammary carcinoma EMT-6 to alkylating agents was demonstrated in the present study to be associated with multiple biochemical changes. These included an up to 1.5-fold increase in activity of phase II drug metabolizing enzymes (DMEs), such as glutathione (GSH), glutathione reductase (GR), glutathione S-transferase (GST), glutathione peroxidase (GPX) and aldehyde dehydrogenase (ALDH), and an up to 88% decrease of phase I DME activity [7-ethoxycumarin O-deethylase (ECOD), P450 reductase (PR)] in the resistant tumors compared with the parental tumor. Transplant of either parental or resistant tumors to mice was accompanied by a decrease of both phase I and phase II DME activity in the livers of female Balb/C mice compared with the non-tumor mice. Moreover, at the protein level, while cytochrome P450 (CYP) IIB1/2 in the liver of mouse bearing both the sensitive and the resistant tumor was significantly diminished compared to that in the liver of non-tumor control mouse in Western analysis, there was actually an increase of this protein in the liver of the host bearing either of the two resistant tumors compared to that of the sensitive tumor-bearing animal. Although this in vivo resistance phenotype is not expressed in cell culture, the profile of most of the enzyme changes in the resistant tumors remained similar in in vitro culture of the isolated tumor cells. Collectively, these results demonstrate that this in vivo alkylating agent resistance is associated with multiple changes of both phase I and phase II DMEs in the resistant tumors, and some of these, such as CYP IIB1/2 protein are further altered in the resistant tumor-bearing mouse liver, suggesting a potential role of systemic factors in this resistance phenotype.
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PMID:Biochemical characterization of in vivo alkylating agent resistance of a murine EMT-6 mammary carcinoma. Implication for systemic involvement in the resistance phenotype. 992 73


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