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1.
Shin-ichi Kuwahara Tsuneo Omura 《Biochemical and biophysical research communications》1980,96(4):1562-1568
The role of cytochrome b5 in the NADPH-supported O-deethylation of p-nitrophenetole catalyzed by cytochrome P-450 was studied with reconstituted systems using two types of cytochrome P-450 (P-450PB and P-450MC) purified from rat liver microsomes. The O-deethylation by P-450PB absolutely required the presence of cytochrome b5, whereas the same reaction catalyzed by P-450MC did not require cytochrome b5. These effects of cytochrome b5 on the activities of reconstituted systems were confirmed by the use of antibodies to cytochrome b5. On the other hand, the oxidations of ethylmorphine and aniline by these two types of cytochrome P-450 did not show significant dependence on cytochrome b5. These observations suggest that the requirement for cytochrome b5 in NADPH-supported drug oxidations depends not only on the species of cytochrome P-450 catalyzing the reactions, but also on the substrates oxidized. 相似文献
2.
Masayuki Katagiri Katsuko Suhara Masahiro Shiroo Yoshiyuki Fujimura 《Biochemical and biophysical research communications》1982,108(1):379-384
The cytochrome P-450 (P-450sccII) and its reductase, NADPH-cytochrome reductase [EC 1.6.2.4], associated with conversion of progesterone to 4-androstene-3,17-dione, were extensively purified from pig testis microsomes. Higher lyase activity (turnover number of 15 mol of the product formed/min/mol of P-450) could be restored by mixing the P-450sccII, its reductase, pig liver cytochrome b5 and cytochrome b5-reductase [EC 1.6.2.2], and phospholipid in the presence of NADPH, NADH, and O2. Omission of either cytochrome b5 or NADH resulted in a significant loss of the lyase activity indicating actual participation of cytochrome b5 in this P-450-mediated steroidogenic system in the testis. 相似文献
3.
《Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism》1985,833(1):151-160
We have reported (Kominami S., Shinzawa K. and Takemori S. (1982) Biochem. Biophys. Res. Commun. 109, 916–921) that a cytochrome P-450 purified from guinea pig adrenal microsomes shows 17α-hydroxylase and C-17,20-lyase activities in a reconstituted system with NADPH-cytochrome P-450 reductase. The homogeneity of the purified cytochrome P-450 was examined with the following methods: isoelectric focusing, immunoelectrophoresis and affinity chromatography on cytochrome b5-immobilized Sepharose. It was found that progesterone competitively inhibited C-17,20-lyase reaction and that progesterone was converted into androstenedione by 17α-hydroxylation followed by the lyase reaction. These results indicate that the dual activities are carried out by a single enzyme (P-45017α,lyase). P-45017α,lyase had the maximum activity at pH 6.1 both for 17α-hydroxylation (6.0 nmol/min per nmol of P-450) and the lyase reaction (11.0 nmol/min per nmol of P-450). Upon addition of cytochrome b5 to the reconstituted system, the optimal pH for 17α-hydroxylation was shifted to 7.0 and that of the lyase reaction to 6.6. The maximum activities at these optimal pH values were almost the same in the presence or absence of cytochrome b5. With the addition of cytochrome b5, both the activities were stimulated above pH 6.3–6.5 and were suppressed below pH 6.3–6.5. These results indicate that cytochrome b5 plays some important role in controlling the dual activities of P-45017α,lyase. 相似文献
4.
Annika Brunström Magnus Ingelman-Sundberg 《Biochemical and biophysical research communications》1980,95(1):431-439
The roles of rabbit liver cytochrome b5, epoxide hydrase and various forms of cytochrome P-450 in the NADPH-dependent metabolism of benzo(a)pyrene were examined. After incorporation of the purified enzymes into phospholipid vesicles, using the cholate gel filtration technique, the various types of cytochrome P-450 did exhibit different stereospecificities in the oxygenation of the substrate. Cytochrome P-450LM2 was found to efficiently convert benzo(a)pyrene in the presence of epoxide hydrase to 4,5-dihydroxy-4,5-dihydrobenzo(a)pyrene whereas cytochrome P-450LM4 primarily participated in the formation of 9,10-dihydroxy-9,10-dihydrobenzo(a)pyrene. By contrast, benzo(a)pyrene was not metabolized by cytochrome P-450LM3. Cytochrome b5 enhanced cytochrome P-450LM2-catalyzed oxygenations 5-fold, whereas cytochrome P-450LM4-dependent oxygenations proceeded at a 3 times higher rate when cytochrome b5 was present in the membrane. 相似文献
5.
Cytochrome b5 purified from neonatal pig testis and that from pig liver stimulated C21 steroid side-chain cleavage (progesterone → androstenedione) catalyzed in vitro by purified cytochrome P-450 from neonatal pig testicular microsomes. Km of testicular cytochrome b5 for the P-450 is 6.3–9.1 × 10?8M and the ability of b5 to stimulate C21 side-chain cleavage is different for cytochromes b5 prepared from different sources. 相似文献
6.
A partially purified preparation of hepatic cytochrome P-448 from 3-methylcholanthrene treated rats was used to produce antisera in rabbits. Using both Ouchterlony double diffusion and quantitative immunoprecipitation analysis, this antisera was found to be more specific for cytochrome P-448 than for cytochrome P-450 from phenobarbital induced rats. The antisera did not form precipitin bands with the following rat liver microsomal proteins: cytochrome b5, NADH-cytochrome b5 reductase, NADPH-cytochrome c reductase or epoxide hydrase. 相似文献
7.
Platelet microsomes were shown to contain cytochromes P-450 and b5 and their respective reductases, NADPH-cytochrome c reductase and NADH-cytochrome b5 reductase. Metyrapone and carbon monoxide (CO), two inhibitors of cytochrome P-450, inhibited both the arachidonic acid-induced platelet aggregation and the formation of aggregating factors from arachidonic acid by isolated microsomes. In addition metyrapone produced a type II spectral change with platelet microsomal cytochrome P-450. The data suggest that cytochrome P-450 may play a role in the complex enzyme systems which convert arachidonic acid to the platelet aggregating factors, cyclic endoperoxides and thromboxane A2. 相似文献
8.
Addition of -nitroanisole to a reaction mixture containing phenobarbital-pretreated rabbit liver microsomes brings about an increase the reoxidation rate of NADH-reduced cytochrome b5. Addition of partially purified cytochrome b5 to a solution containing microsomes results in a marked increase in both NADH- and NADPH-dependent O-demethylation of -nitroanisole. -Nitroanisole also increases the rate of NADH mediated cytochrome P-450 reduction. From these and other results described in the Discussion section, we confirm that electrons required for NADH-dependent O-demethylation of -nitroanisole is transfered from NADH to cytochrome P-450 via cytochrome b5 and that cytochrome P-450 is the enzyme which catalyzes -nitroanisole O-demethylation. 相似文献
9.
Y Yoshida Y Aoyama H Kumaoka S Kubota 《Biochemical and biophysical research communications》1977,78(3):1005-1010
Cytochrome P-450 was purified from microsomes of anaerobically grown yeast to a specific content of 12–15 nmoles per mg of protein with a yield of 10–30%. Upon sodium dodecylsulfate/polyacrylamide gel electrophoresis, the purified preparation yielded a major protein band having a molecular weight of about 51,000 together with a few faint bands. It was free from cytochrome b5, NADH-cytochrome b5 reductase, and NADPH-cytochrome c (P-450) reductase. In the oxidized state it exhibited a low-spin type absorption spectrum, and its reduced CO complex showed a Soret peak at 447–448 nm. It was reducible by NADPH in the presence of an NADPH-cytochrome c reductase preparation purified from yeast microsomes. Its conversion to the cytochrome P-420 form was much slower than that of hepatic cytochrome P-450. 相似文献
10.
Mitsukazu Kitada Chiyo Yamazaki Kouzi Hirota Haruo Kitagawa 《Biochemical and biophysical research communications》1980,93(4):1020-1026
Cytochrome P-450 was purified from phenobarbital-treated guinea pigs to a specific content of 19.8 nmoles per mg of protein, and was free of cytochrome b5 and NADPH-cytochrome reductase. The purified cytochrome P-450 gave a single protein band on sodium dodecylsulfate-polyacrylamide gel electrophoresis, and an apparent molecular weight of about 49,000 was estimated. Benzphetamine N-demethylation activity could be reconstituted by mixing the purified cytochrome, NADPH-cytochrome reductase and phosphatidylcholine. 相似文献
11.
The role of cytochrome 5 in the -nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome 5. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome 5 reductase, and Triton X-100 in addition to cytochrome 5. The omission of cytochrome 5 from the complete system entirely abolished the activity. These results clearly show that cytochrome 5 is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome 5 in such a way that the second electron is transferred from cytochrome 5 and thus exhibits the demethylase activity. 相似文献
12.
Bengt Jernström Jorge Capdevila Sten Jakobsson Sten Orrenius 《Biochemical and biophysical research communications》1975,64(3):814-822
Cytochrome P-450 from rat lung microsomes has been solubilized and purified 8-fold by using affinity chromatography on an ω-amino--octyl derivative of Sepharose 4B. The purified fraction was free of cytochrome 5 and NADPH-cytochrome reductase and showed spectral characteristics similar to those of lung microsomal cytochrome P-450. When combined with NADPH-cytochrome reductase partially purified from liver microsomes, the cytochrome P-450 fraction supported the hydroxylation of benzo (α)pyrene and the activity was proportional to the content of the hemoprotein. No absolute requirement for phosphatidylcholine was found. 相似文献
13.
Summary Measurement of the effect of drugs on the in vivo rates of synthesis of rabbit liver organelle bound proteins were measured following individual treatments with the inducers phenobarbital, 3-methylcholanthrene and PCB (a mixture of polychlorinated biphenyls) and the inhibitors, cycloheximide, aflatoxin 13, chloramphenicol and actinomycin D. Following their isolation from a homogenate containing the combined livers of 14C-leucine injected experimental animals and 3H-leucine injected control animals, purified fractions of the following proteins were prepared: microsomal cytochrome b5, cytochrome P-450, NADH-cytochrome b5 reductase, NADPH-cytochrome P-450 reductase and proteolipids, outer mitochrondrial membrane cytochrome b5, NADH-cytochrome b5 reductase and proteolipids, inner mitochrondrial membrane cytochrome c, NADH dehydrogenase and proteolipids, intermitochrondrial membrane cytochrome b5 and circulating serum albumin.This research was supported by grants from the PSC-BHE Research Award Program of The City University of New York, US Public Health Service Grant SO 7 PRO7132-07 and the Alma Toorock Fund for Cancer Research. 相似文献
14.
Cytochrome b5 as electron donor to rabbit liver cytochrome P-450LM2 in reconstituted phospholipid vesicles 总被引:3,自引:0,他引:3
M Ingelman-Sundberg I Johansson 《Biochemical and biophysical research communications》1980,97(2):582-586
When incorporated into phospholipid vesicles containing NADPH-cytochrome P-450 reductase and P-450LM2, cytochrome b5 enhanced the rate of NADPH-supported hydroxylation of 7-ethoxycoumarin or -nitroanisole about 5-fold. Cytochrome b5 did not affect the rate of NADPH-oxidation, nor the rate of NADPH-supported formation of the ferrous CO-complex of cytochrome P-450. However, the cytochrome b5-mediated increase in product formation was found to be correlated with concomitant decreases in the production of H2O2 or in the system, thus strongly indicating cytochrome b5 being a more efficient donor of the second electron to cytochrome P-450 than is NADPH-cytochrome P-450 reductase. 相似文献
15.
David R. Dutton Susan K. McMillen Andrew Parkinson 《Journal of biochemical and molecular toxicology》1988,3(2):131-145
Sodium cholate, Emulgen 911, and (3-[(-cholamidopropyl)-dimethyl- ammonio]-1-propanesulfonate) (CHAPS) were selected to examine the effects of ionic, nonionic, and zwitterionic detergents on testosterone hydroxylation catalyzed by four purified isozymes of rat liver microsomal cytochrome P-450, namely P-450a, P-450b, P-450c, and P-450h, in reconstituted systems containing optimal amounts of dilauroylphosphatidylcholine and saturating amounts of NADPH- cytochrome P-450 reductase (reductase). The major phenobarbital-inducible form of rat liver microsomal cytochrome P-450, designated P-450b, was extremely sensitive to the inhibitory effects of Emulgen 911, which is used in several procedures to purify this and other forms of cytochrome P-450. In contrast, sodium cholate and CHAPS had little effect on the catalytic activity of cytochrome P-450b, even at ten times the concentration of Emulgen 911 effecting 50% inhibition (IC-50). By substituting the zwitterionic detergent CHAPS for Emulgen 911, we purified cytochrome P-450b without the use of nonionic detergent. The protein is designated cytochrome P-450b* to distinguish it from cytochrome P-450b purified with the use of Emulgen 911. NADPH-cytochrome P-450 reductase was also purified both with and without the use of nonionic detergent. The absolute spectra of cytochrome P-450b and P-450b* were indistinguishable, as were the carbon monoxide (CO)- and metyrapone-difference spectra of the dithionite-reduced hemoproteins. When reconstituted with NADPH-cytochrome P-450 reductase and dilauroylphosphatidylcholine, cytochromes P-450b and P-450b* catalyzed the N-demethylation of benzphetamine and aminopyrine, the 4-hydroxylation of aniline, the O-dealkylation of 7-ethoxycoumarin, the 3-hydroxylation of hexobarbital, and the 6-hydroxylation of zoxazolamine. Both hemo-proteins catalyzed the 16α- and 16β-hydroxylation of testosterone, as well as the 17-oxidation of testosterone to androstenedione. Both hemoproteins were poor catalysts of erythromycin demethylation and benzo[a]pyrene 3-/9-hydroxylation. The rate of biotransformation catalyzed by cytochrome P-450b* was up to 50% greater than the rate catalyzed by cytochrome P-450b when reconstituted with either reductase or reductase*. The activity of cytochrome P-450b and P-450b* increased up to 50% when reconstituted with reductase* instead of reductase. In addition to establishing the feasibility of purifying an isozyme of rat liver microsomal cytochrome P-450 without the use of nonionic detergent, these results indicate that the catalytic activity of cytochrome P-450 is not unduly compromised by residual contamination with the nonionic detergent Emulgen 911. 相似文献
16.
Purification and properties of P-450LM3b, a constitutive form of cytochrome P-450, from rabbit liver microsomes 总被引:3,自引:0,他引:3
This laboratory has previously reported the occurrence in rabbit liver microsomes of a non-inducible form of cytochrome P-450, designated P-450lm3b because of its electrophoretic mobility relative to that of phenobarbital-inducible P-450lm2 and 5,6-benzoflavone-inducible P-450lm4. In the present study, P-450lm3b was purified to electrophoretic homogeneity and a specific content of over 19 nmol per mg of protein by chromatographic procedures carried out in the presence of detergents. The isolated cytochrome has a minimal molecular weight of 52,000 and exhibits absorption maxima at 418, 537, and 571 nm in the oxidized state, 412 and 547 nm in the reduced state, and 451 and 555 nm as the CO complex. In a reconstituted system containing NADPH-cytochrome P-450 reductase and phosphatidylcholine, P-450lm3b has relatively high activity in the hydroxylation of testosterone in the 6β and 16α positions as well as significant activity toward a number of other substrates tested. The NADPH oxidase activity of P-450lm3b is less than half that of P-450lm2 and lm4. 相似文献
17.
The purpose of this report is to review the current literature on cytochrome 5 in hepatic microsomes and to draw conclusions as to its role in microsomal electron transfer pathways. For details concerning the history of cytochrome 5 the reader is reffered to reviews by C. F. Strittmatter (1) and P. Strittmatter (2). For information on the chemistry of cytochrome 5 the reader is reffered to the papers by Ozols and Strittmatter (3), Kajihara and Hagihara (4), and Ehrenberg and Bois-Poltoratsky (5). For more recent studies on the isolation and properties of detergent solubilized cytochrome 5, which contains a hydrophobic peptide enabling reincorporation into membranes, the reader is referred to references 6-12.For simplicity, this minireview is divided into four parts, reflecting areas of study on the role of cytochrome 5 in the microsomes. One major area is in fatty acid 9 desaturation. Two other areas concern cytochrome 5 involvement in cytochrome P-450 mediated mixed function oxidations. The fourth section deals with other non-cytochrome P-450 pathways in which cytochrome 5 is suggested as being a component. 相似文献
18.
Partial purification and properties of cytochromes P-450 and P-448 from rat liver microsomes 总被引:2,自引:0,他引:2
W Levin A Y Lu D Ryan S West R Kuntzman A H Conney 《Archives of biochemistry and biophysics》1972,153(2):543-553
Cytochromes P-450 and P-448 in rat liver microsomes were solubilized with sodium cholate and were partially purified. The preparations contained 5.0–5.5 nmoles of cytochrome P-450 or P-448 per mg of protein; contamination with cytochrome P-420 and cytochrome b5, was less than 10% of the total heme content. The absolute spectra of Cytochromes P-450 and P-448 differed only slightly; both hemoproteins had a Soret peak at 418–419 nm in the oxidized absolute spectra and at 448 and 450 nm in the reduced plus CO absolute spectra. Both hemoproteins showed typical type I (benzphetamine) and type II (aniline) binding spectra but differed in their binding of hexobarbital (another type I substrate). The total phospholipid content of the preparation (per mg protein) has been reduced by approximately 90% relative to microsomes and the hemoprotein has been purified 20–25 fold with respect to phospholipid. The partially purified hemoprotein fractions, after combination with a reductase and lipid fraction, were capable of oxidizing a variety of substrates inluding drugs, steroids, and chemical carcinogens. 相似文献
19.
Both the cytochrome b5 level and NADH cytochrome b5 reductase activity in rat liver microsomes were increased 2-fold by repeated i.p. administration of 1.5 mmol/kg propylthiouracil (PTU) for 2 weeks, but neither the cytochrome P-450 level nor NADPH cytochrome P-450 reductase activity were affected by the treatment. Liver microsomes from PTU-treated rats showed a significant decrease in aminopyrine N-demethylation, but not in benzphetamine N-demethylation, aniline hydroxylation or 7-ethoxycoumarin O-deethylation. A single administration of the compound had no effect on any components of the system. , drug hydroxylation activities were not affected by PTU up to 1.0 mM. From the above evidence, repeated administration of PTU selectively induced cytochrome b5 and NADH cytochrome b5 reductase in rat liver microsomes. 相似文献
20.
We have shown earlier that microsomal cytochrome b
5 can form a specific complex with mitochondrial cytochrome P450 (cytochrome P450scc). The formation of the complex between these two heme proteins was proved spectrophotometrically, by affinity chromatography on immobilized cytochrome b
5, and by measuring the cholesterol side-chain cleavage activity of cytochrome P450scc in a reconstituted system in the presence of cytochrome b
5. To further study the mechanism of interaction of these heme proteins and evaluate the role of negatively charged amino acid residues Glu42, Glu48, and Asp65 of cytochrome b
5, which are located at the site responsible for interaction with electron transfer partners, we used sitedirected mutagenesis to replace residues Glu42 and Glu48 with lysine and residue Asp65 with alanine. The resulting mutant forms of cytochrome b
5 were expressed in E. coli, and full-length and truncated forms (shortened from the C-terminal sequence due to cleavage of 40 amino acid residues) of these cytochrome b
5 mutants were purified. Addition of the truncated forms of cytochrome b
5 (which do not contain the hydrophobic C-terminal sequence responsible for interaction with the membrane) to the reconstituted system containing cytochrome P450scc caused practically no stimulation of catalytic activity, indicating an important role of the hydrophobic fragment of cytochrome b
5 in its interaction with cytochrome P450scc. However, full-length cytochrome b
5 and the full-length Glu48Lys and Asp65Ala mutant forms of cytochrome b
5 stimulated the cholesterol side-chain cleavage reaction catalyzed by cytochrome P450scc by 100%, suggesting that residues Glu48 and Asp65 of cytochrome b
5 are not directly involved in its interaction with cytochrome P450scc. The replacement of Glu42 for lysine, however, made the Glu42Lys mutant form of cytochrome b
5 about 40% less effective in stimulation of the cholesterol side-chain cleavage activity of cytochrome P450scc, indicating that residue Glu42 of cytochrome b
5 is involved in electrostatic interactions with cytochrome P450scc. Residues Glu42 and Glu48 of cytochrome b
5 appear to participate in electrostatic interaction with microsomal type cytochrome P450. 相似文献