UNIPROT:Q8NEX9 - FACTA Search

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Query: UNIPROT:Q8NEX9 (reductase)
26,410 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cytochrome P450 in the mitochondria of the adrenal cortex functions in the monooxygenation reactions for the biosynthesis of various steroid hormones, such as cholesterol side chain cleavage, hydroxylation at 11 beta-position and that at 18-position of the steroid structure. The cytochrome is firmly associated with the mitochondrial membrane and therefore can be isolated only by the aid of ionic or non-ionic detergent. Recently, two cytochromes P450 each catalyzing a specified reaction have been purified to a homogeneous state, that is, P450scc having cholesterol side chain cleavage activity and P45011 beta having 11 beta-hydroxylation activity. The properties of these purified P450's as well as the other components of the monooxygenase system, adrenodoxin and adrenodoxin reductase, are, therefore, summarized and compared to those of P450 in the mitochondrial preparation in situ. Among many findings, both purified cytochromes P450 were revealed to be a low-spin type hemoprotein and their spin states were changed to a high-spin state by being complexed with the corresponding substrate. The binding of a substrate also facilitated the reduction of the cytochrome and appeared to increase the stability of the oxygenated form of cytochrome P450. These effects are important from the point of view that the primary role of the heme of cytochrome P450 is the activation of molecular oxygen. In addition, the results of our detailed kinetic studies on the transfer of electrons from adrenodoxin to cytochrome P450 in the reconstituted system have also been described. Finally, the topology of adrenodoxin and the reductase were shown to be on the inner mitochondrial membrane by a peroxidase-labeled antibody method.
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PMID:Cytochrome P450 in adrenocortical mitochondria. 22 25

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.
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PMID:Solubilization and partial characterization of rat liver squalene epoxidase. 23 59

A protein (designated as protein-B) was purified from liver microsomes of adult male guinea pigs by an affinity chromatography with omega-aminooctyl Sepharose 4B, followed by HPLC using DEAE-5PW and hydroxyapatite columns which had been used to purify a cytochrome P450 (P450) isozyme (P450-A) from the same subcellular fraction (Narimatsu et al., Biochem Biophys Res Commun 172: 607-613, 1990). Protein-B had a molecular mass of 49 kDa in SDS-PAGE, but did not show absorbance at 417 nm for heme. Further, it did not show any oxidative activities towards aniline (AN), d-benzphetamine (d-BP), p-nitroanisole (p-NA) or delta 9-tetrahydrocannabinol (delta 9-THC) in a reconstituted system including dilauroylphosphatidylcholine, NADPH-P450 reductase, and cytochrome b5. However, antiserum against protein-B raised in rabbits suppressed liver microsomal oxidative activities towards d-BP and p-NA dose-dependently. The antibody decreased delta 9-THC oxidative activity most effectively, but did not decrease AN hydroxylation activity. Antiserum against P450-A suppressed all the activities towards these four substrates, especially towards delta 9-THC, in liver microsomes of male guinea pigs. Moreover, reconstitution with hemin made it possible for protein-B to produce some oxidative activity toward delta 9-THC. These results suggest that protein-B is also a cytochrome P450 isozyme which has lost a heme moiety during purification steps. Both P450-A and protein-B could have a role as cytochrome P450 isozymes in the oxidative metabolism of drugs, especially that of delta 9-THC by the liver microsomes of adult male guinea pigs.
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PMID:Immunochemical characterization of a cytochrome P450 isozyme and a protein purified from liver microsomes of male guinea pigs and their roles in the oxidative metabolism of delta 9-tetrahydrocannabinol by guinea pig liver microsomes. 131 42

Attempts to covalently link NADPH-cytochrome P450 reductase to cytochrome P450 2B4 using a water-soluble carbodiimide, 1-ethyl-3-(3-dimethylisopropyl)carbodiimide, were unsuccessful, despite the fact that under the same conditions about 30% of P450 2B4 could be covalently linked with cytochrome b5 in a functionally active complex (Tamburini, P. P., and Schenkman, J. B. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 11-15). This suggested that the functional electron transfer complex between P450 2B4 and reductase is not stabilized by electrostatic forces. Raising the ionic strength of the medium is disruptive to salt bridges and was used to further test whether P450 2B4 and the reductase form charge-pairing complexes. Instead of inhibiting electron transfer, high ionic strength increased the apparent fast phase rate constant and the fraction of P450 2B4 reduced in the fast phase. The possibility that electron transfer between NADPH-cytochrome P450 reductase and P450 2B4 is diminished by charge repulsion was examined. Consistent with this hypothesis, the Km of P450 2B4 for reductase was decreased 26-fold by increasing the ionic strength from 10 to 100 mM sodium phosphate without affecting the Vmax. The rate of benzphetamine N-demethylation also was increased by elevation of the ionic strength. Electron transfer from the reductase to other charged redox acceptors, e.g. cytochrome c and ferricyanide, was also stimulated by increased ionic strength. However, no similar stimulation was observed with the uncharged acceptor 1,4-benzoquinone. Polylysine, a polypeptide that binds to anionic sites, enhanced electron transfer from NADPH to ferricyanide and the apparent fast phase of reduction of cytochrome P450. The results are consistent with the hypothesis that charges on NADPH-cytochrome P450 reductase and cytochrome P450 decrease the stability of the electron transfer complex.
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PMID:The cytochrome P450 2B4-NADPH cytochrome P450 reductase electron transfer complex is not formed by charge-pairing. 132 14

The kinetics of NADPH-dependent reduction of cytochrome P450 LM2 in the soluble monomeric reconstituted system in the absence of any substrate is shown to be monophasic. We show that ferrous cytochrome c acts as a competitive inhibitor of the reduction. In the presence of 1 mM benzphetamine an additional extremely fast phase was observed. Under these conditions ferrous cytochrome c was found to be a competitive inhibitor of the slow phase of the reduction process, which accounted for 80% of the total reduction amplitude. Inhibition experiments yield a dissociation constant for the LM2-reductase complex of 3.0 +/- 1.5 microM. This constant was the same both in the presence and in the absence of benzphetamine. Based on these data we conclude that cytochromes P450 and c bind to the same center on the NADPH-cytochrome P450 reductase molecule. Comparative analysis of the amino acid sequences reveals a detectable similarity between cytochrome c and cytochrome P450 LM2 at positions 68-87 and 121-145, respectively. In addition, a substantial similarity was shown for sequence fragments 204-224 of NADPH-cytochrome P450 reductase and 40-60 of cytochrome b5. Based on these findings a hypothesis for the location of the centers of intermolecular interactions on the molecules of cytochrome P450 LM2 and NADPH-cytochrome P450 reductase is proposed.
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PMID:Cytochrome C (Fe2+) as a competitive inhibitor of NADPH-dependent reduction of cytochrome P450 LM2: locating protein-protein interaction sites in microsomal electron carriers. 132 42

The hamster, a rodent possessing adrenal 17 alpha-hydroxylase activity, was used to study the effect of ACTH on the regulation of cortisol formation in vivo. The characterization of the mRNA and protein of hamster adrenal steroidogenic enzymes revealed close similarities between this animal and other mammalian species. The hamster adrenal RNA hybridized in a single band to cDNA probes for bovine adrenal P450scc, P450(17 alpha), P450c21, to mouse adrenal P450(11 beta), and to pig testis 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) in the areas of 2.2, 2.0, 2.3, 2.0, and 2.1 kilobases, respectively. Immunoblotting analyses revealed the presence of single protein bands reacting with antibodies to bovine P450scc, P450c21, porcine P450(17 alpha), or human placental 3 beta HSD in the areas of 52, 55, 51, and 41 kilodaltons, respectively, whereas two protein bands were detected at 48 and 52 kilodaltons with the antibody to bovine P450(11 beta). After stimulation with ACTH injected at 5-h intervals over 20 h, plasma cortisol levels, which were already increased 2.5 h after the first injection, remained elevated for the duration of treatment and returned to control values 15 h after the last injection. The ratios of plasma cortisol to corticosterone were 1.5, 3.9, and 7 at 0, 2.5, and 5 h after the first injection and continued to rise to a value of 11 at 15 h after multiple injections. This ratio returned to control values 15 h after cessation of either the short term (one injection) or long term (five injections) treatment, indicating a control mechanism favoring cortisol formation upon ACTH stimulation. Of the adrenal enzyme systems examined, only three were directly affected by ACTH treatment. The mRNA level of 3-hydroxy-3-methylglutaryl-coenzyme-A reductase, the key precholesterol regulatory step, increased after ACTH administration within 2.5 h and remained elevated during the entire study period. ACTH provoked a rapid and sustained increase in P450scc mRNA levels, which decreased very slowly after cessation of treatment without reaching control values 30 h after the last injection. Meanwhile, ACTH treatment caused no changes in the amount of adrenal cytochrome P450scc protein during treatment and 30 h after its cessation. Therefore, we postulate that factors other than newly synthesized P450scc protein participate in the control of this rate-limiting step. The high P450scc mRNA levels observed suggest stabilization of mRNA and posttranscriptional events affecting its catabolism.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:In vivo effects of adrenocorticotropin on hamster adrenal steroidogenic enzymes. 132 21

In contrast to the increase in reaction rate of microsomal NADPH-cytochrome-P450 reductase activity resulting from low-level microwave perturbation (reported earlier) transformations involving the entire MFO-pathway were inhibited by a microwave field. Dealkylation of 7-ethoxycoumarin was inhibited 25% and demethylation of p-nitroanisole was inhibited 40% when the reaction was carried forward in a 9.14 GHz CW field. Microsomal preparations from the liver of mature chickens had enzymic characteristics (kinetic constants, inhibitor-response spectrum) for these substrates similar to those reported for rodent and human MFO complex.
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PMID:Mixed function oxidase demethylase and dealkylase activity in an electromagnetic field. 134 46

Mitochondrial ferredoxins mediate electron transfer from NADPH:ferredoxin oxidoreductase to cytochrome P450 enzymes. Previous studies on human ferredoxin, in which acidic residues were replaced with neutral amino acids, established that Asp-76 and Asp-79 are are important for binding to both reductase and P450 (Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612). Here we report that replacement of Asp----Glu at position 76 or 79, whereas maintaining negative charge at these positions also results in dramatic decreases in binding affinity for both electron transfer partners (5-100-fold, delta(delta G) approximately 1.0-2.8 kcal/mol). These results imply that the active electron transfer complexes in these systems are dominated by a stable form which requires specific pairwise electrostatic interactions of fixed geometry for recognition and binding. This mechanism contrasts with that proposed for other electron transfer systems (as exemplified by cytochrome c) in which electrostatic interactions are believed to function primarily in precollisional orientation leading to "encounter complexes" having multiple geometries of similar free energy.
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PMID:Electrostatic interactions stabilizing ferredoxin electron transfer complexes. Disruption by "conservative" mutations. 134 3

Cytochrome P-450 2E1 is induced in adult rabbits by treatment with alcohol, imidazole, and a variety of other agents, as shown earlier in this laboratory, but it is not known whether the highly homologous P-450 2E2 is similarly induced. In this study, the effects of imidazole on 2E2 expression were examined in neonatal rabbits, in which 2E1 is not detectable. Treatment of the animals with imidazole on days 8 through 11 after birth caused a 3-fold increase in the content of total P-450 in liver microsomes. In contrast, the microsomal content of cytochrome b5 and NADPH-P450 reductase was not changed. Immunoblot analysis revealed a significant increase in the level of P-450 2E2 (3-fold) as well as 1A1 (> 10-fold) and 1A2 (> 2-fold) in hepatic microsomes from imidazole-treated neonatal rabbits. The rates of microsomal N-demethylation of N-nitrosodimethylamine and O-deethylation of 7-ethoxyresorufin were similarly increased from 1.3 and 0.03 nmol/min/mg protein, respectively, to 5.6 and 0.24 nmol/min/mg protein, respectively, by imidazole treatment. Blot analysis indicated that the levels of 2E2, 1A1, and 1A2 mRNAs are not increased by imidazole treatment and that 2E1 mRNA is not detectable in either untreated or imidazole-treated neonates. The induction of P-450 2E2 was confirmed by NH2-terminal amino acid sequence analysis of immunopurified 2E protein from hepatic microsomes of imidazole-treated neonatal rabbits.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Induction of P-450 cytochromes 2E2, 1A1, and 1A2 by imidazole in neonatal rabbits. 136 28

The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an assay involving high performance liquid chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (M(r) = 50,000). The NH2-terminal amino acid sequence is as follows: Val-Leu-Trp-Gly-Leu-Leu-Gly-Ala-Leu-Leu-Met-Val-Met-Val-Gly-, which is different from that of any other P450s so far reported. The specific content of the enzyme was 13.3 nmol of cytochrome P450/mg of protein. Upon reconstitution with NADPH-cytochrome P450 reductase and cytochrome b5, the P450 enzyme showed a high activity of 12 alpha-hydroxylation with a turnover number of 36.6 min-1 at 37 degrees C. The omission of either cytochrome P450 or NADPH-cytochrome P450 reductase resulted in complete loss of activity, and the omission of cytochrome b5 resulted in 40% loss of activity. Antibodies prepared from mouse inhibited the 12 alpha-hydroxylase activity of rabbit liver microsomes about 90% and that of the rat liver microsomes 50%. The enzyme activity was not inhibited by other antibodies raised against other forms of P450 that catalyze different monooxygenation reactions toward xenobiotics or endogenous substrates. Anti-cytochrome b5 antibody inhibited the activity 40%, suggesting the functional role of this protein, and anti-reductase inhibited the activity almost completely. The microsomal enzyme activity was markedly elevated by starvation or streptozotocin administration to the animals. However, an immunoblotting experiment showed no correlation between the enzyme activity and the amount of protein, suggesting that post-translational modification may occur.
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PMID:Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase. 140 Apr 44

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