首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献,搜索用时 15 毫秒
1.
Cell-free extracts from sonically disrupted Bacillus megaterium ATCC 13368 hydroxylated a variety of 3-oxo-delta4-steroids in position 15beta in the presence of NADPH and O2. Ring A-reduced, aromatic and 3beta-hydroxy-delta5-steroids did not serve as substrates for the 15beta-hydroxylase system. Using ion exchange chromatography on DEAE-cellulose and gel filtration on Ultrogel ACA-54 it was possible to resolve the hydroxylase system into three proteins: a strictly NADPH-dependent FMN-containing (megaredoxin reductase), an iron-sulfur protein (megaredoxin), and cytochrome P-450 (P-450meg). The activity of the 15beta-hydroxylase system was fully reconstituted upon combination of these three proteins and addition of NADPH. Megaredoxin had an apparent sulfur to iron ration of 0.98 and showed g-signals at 1.90, 1.93, and 2.06 when analyzed by electron paramagnetic reso0 times and the preparation contained 1 to 2 nmol of cytochrome P-450 per mg of protein. This preparation of cytochrome P-450meg sedimented as a homogeneous zone on sucrose gradients with a sedimentation coefficient of 3.3 S and contained 0.94 nmol of heme per nmol of cytochrome P-450. The oxidized form of cytochrome P-450meg showed absolute absorption maxima at 416, 528, and 565 nm whereas the reduced form showed maxima at 411 and 542 nm. The following scheme is suggested for the electron transport in the 15beta-hydroxylase system in B. megaterium: NADPH leads to megaredoxin reductase leads to megaredoxin leads to cytochrome P-450meg.  相似文献   

2.
Studies were conducted to further examine the mechanisms responsible for gonadal hormone effects on the rat adrenocortical 11beta-hydroxylase system. Despite higher concentrations of cytochrome P-450 and larger 11-deoxycorticosterone (DOC)-induced difference spectra in adrenal mitochondria from females than males, no sex difference in 11beta-hydroxylase activity was observed. The pregnenolone-induced difference spectrum, indicative of cholesterol binding to cytochrome P-450, also was similar in males and females. Testosterone administration to castrated males lowered both 11beta-hydroxylase activity and mitochondrial cytochrome P-450 content. Estradiol produced the opposite effects in castrated females. However, when given to ACTH-replaced hypophysectomized rats, neither testosterone nor estradiol affected cytochrome P-450 levels or the rate of 11beta-hydroxylation. These observations, taken with the known effects of estradiol and testosterone on ACTH secretion in rats and the effects of ACTH on 11beta-hydroxylation, indicate that gonadal hormone effects on the 11beta-hydroxylase system are mediated by ACTH.  相似文献   

3.
Mechanisms of cytochrome P-450 catalysis   总被引:8,自引:0,他引:8  
Cytochrome P-450 (P-450) enzymes catalyze the oxidation of a wide variety of substrates. Although a large number of P-450s have been characterized in different species and tissues, the mechanisms of catalysis of oxygenation may be understood in terms of a few basic principles. The chemistry is dominated by the ability of a high-valent formal (FeO)3+ species to carry out one-electron oxidations through the abstraction of hydrogen atoms, abstraction of electrons in n or pi orbitals, or the addition to pi bonds. A series of radical recombination reactions then completes the oxidation process. The protein structures are postulated to provide the axial thiolate ligand to the heme, to control the juxtaposition of the substrate (and therefore the regio- and stereoselectivity of oxidation), to alter the effective oxidation potential of the (FeO)3+ complex, and possibly to participate in specific acid/base catalysis in the oxidation of some substrates.  相似文献   

4.
The synthesis and maturation of the precursor forms of three mitochondrial enzymes involved in steroid hormone biosynthesis have been studied in vivo. Primary cultures of bovine adrenocortical cells were radiolabeled with [35S] methionine and newly synthesized cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 11 beta-hydroxylase cytochrome P-450 (P-450(11)beta), and adrenodoxin immunoisolated using specific antibodies. Both the precursor and mature forms of P-450scc and P-450(11)beta were detected during short periods of pulse labeling; however, the precursor forms were transitory in nature while their corresponding mature forms accumulated. Pulse-chase experiments showed that the precursor form of each cytochrome P-450 had an apparent half-life of 3.5 min. In contrast, the precursor form of adrenodoxin was not readily detected in pulse-labeling experiments until a substantial amount of its mature form had accumulated. When the cultured cells were treated with a chelator of divalent cations (o-phenanthroline) or a mitochondrial uncoupler (dinitrophenol), the maturation of all three precursors was inhibited. The synthesis of the P-450scc and P-450(11)beta precursors was induced in cells maintained in the presence of adrenocorticotropin, and the rates of appearance of their processed forms were also increased. The mature forms of all three proteins were immunoisolated from a trypsinized mitochondrial fraction prepared from the radiolabeled cells, demonstrating that the mature proteins were localized within the organelle. These studies establish that the maturation of the precursor forms of the mitochondrial steroidogenic enzymes are characterized by steps similar to those reported for other mitochondrial precursor proteins.  相似文献   

5.
Three forms of cytochrome P-450, designated as P-450-HM1, P-450-HM2, and P-450-HM3, were isolated from human liver microsomes using high-performance liquid chromatography (HPLC) techniques. Each purified preparation showed a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). From the results of SDS-PAGE, the molecular weights of P-450-HM1, P-450-HM2, and P-450-HM3 were estimated to be 51,000, 54,000, and 52,000, respectively. The oxidized absolute spectra of these three forms of cytochrome P-450 showed Soret absorption peaks at around 417 nm, indicating that these forms were in the low spin state. In a reconstituted system, P-450-HM1 showed the highest catalytic activities of nifedipine and (S)- or (R)-nilvadipine oxidases. The same form showed higher activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. P-450-HM2 showed high N-demethylase activities for benz-phetamine and aminopyrine, and also showed the highest activity of testosterone 16 beta-hydroxylase among the three forms, while it did not show detectable activities of testosterone 6 beta-hydroxylase and progesterone 6 beta- and 16 alpha-hydroxylases. Anti-P-450-HM1 immunoglobulin G (IgG), but not anti-P-450-HM2 IgG, inhibited the activities of testosterone 6 beta-hydroxylase and nifedipine and nilvadipine oxidases in human liver microsomes. Anti-P-450-HM1 IgG was also inhibitory against progesterone 6 beta- and 16 alpha-hydroxylases.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

6.
Cytochrome P-450-dependent digitoxin 12 beta-hydroxylase from cell cultures of foxglove (Digitalis lanata) was solubilized from microsomal membranes with CHAPS (3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulphonic acid). Cytochrome P-450 was separated from NADPH: cytochrome c (P-450) reductase by ion-exchange chromatography on DEAE-Sephacel. NADPH:cytochrome c (P-450) reductase was further purified by affinity chromatography on 2',5'-ADP-Sepharose 4B. This procedure resulted in a 248-fold purification of the enzyme; on SDS/polyacrylamide-gel electrophoresis after silver staining, only one band, corresponding to a molecular mass of 80 kDa, was present. The digitoxin 12 beta-hydroxylase activity could be reconstituted by incubating partially purified cytochrome P-450 and NADPH:cytochrome c (P-450) reductase together with naturally occurring microsomal lipids and flavin nucleotides. This procedure yielded about 10% of the original amount of digitoxin 12 beta-hydroxylase.  相似文献   

7.
Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.  相似文献   

8.
Two forms of cytochrome P-450 (P-450 human-1 and P-450 human-2) have been purified from human liver microsomes to electrophoretic homogeneity. P-450 human-1 and P-450 human-2 differ in their apparent molecular weights (52,000 and 56,000, respectively) and Soret peak maxima in the CO-binding reduced difference spectrum (447.6 and 450.3 nm, respectively). In the reconstituted system using rat liver NADPH-cytochrome c (P-450) reductase, P-450 human-2 more effectively oxidized benzo(a)pyrene (80-fold), ethylmorphine (2-fold), and 7-ethoxycoumarin (2-fold) than did P-450 human-1. However, P-450 human-1 showed higher testosterone 6 beta-hydroxylase activity, and the activity was markedly increased by the inclusion of cytochrome b5 or spermine in the reconstituted system. Antibodies raised against P-450 human-1 inhibited more than 80% of microsomal testosterone 6 beta-hydroxylase activity in human liver. Immunoblotting analysis using anti-P-450 human-1 IgG revealed a single immuno-staining band near Mr 52,000 in all human liver samples examined. The amount of immunochemically determined P-450 human-1 varied in parallel with the testosterone 6 beta-hydroxylase activity in human liver. These results indicate that P-450 human-1 is a major form of cytochrome P-450 responsible for microsomal testosterone 6 beta-hydroxylation. Thus, this paper is the first report on human cytochrome P-450 responsible for testosterone 6 beta-hydroxylation, which is the major hydroxylation pathway in human liver microsomes.  相似文献   

9.
10.
Human cytochrome P-450 enzymes.   总被引:6,自引:0,他引:6  
F P Guengerich 《Life sciences》1992,50(20):1471-1478
Cytochrome P-450 (P-450) enzymes have been studied extensively in experimental animal models and much is known regarding their structures, regulation, and mechanisms of catalysis. In recent years investigations have been extended to the human P-450s. There are more than 30 different characterized human P-450s in the superfamily, and collectively they are probably the most significant enzymes involved in the metabolism of drugs, carcinogens, and steroids. The levels of many of the P-450s and their catalytic activities can vary considerably because of polymorphism, induction, and inhibition. The catalytic specificity of the P-450s can range from being very non-discriminatory to very exacting, and clinical consequences of drugs and steroids can be related to variations in P-450 levels. Defects in the rate-limiting P-450 reactions in steroidogenesis (due to genetic deficiencies) have been shown to be debilitating and even fatal.  相似文献   

11.
Cytochromes P-450 are extremely important in the oxidative metabolism of a variety of endogenous and exogenous compounds in pro- and eukaryotic organisms. Progress in understanding the structure and mechanism of action of this superfamily of enzymes has been hampered by the properties of the eukaryotic enzymes and the availability of only one well-characterized prokaryotic enzyme as a model. We report here the isolation of a Pseudomonas species which will utilize a monoterpene natural product, alpha-terpineol, as its sole source of carbon and energy. Approximately 1% of the soluble protein in the cell-free extract is a novel cytochrome P-450 (P-450terp). This enzyme and its associated iron sulfur protein electron carrier (terpredoxin) have been purified to homogeneity and their NH2-terminal amino acid sequences determined. The amino acid sequences of six tryptic peptide fragments of cytochrome P-450terp have also been determined. This sequence information was used to clone the gene encoding cytochrome P-450terp. Three clones representing approximately 8 kilobase pairs of unique sequences were selected and sequenced. Five non-overlapping open reading frames (ORFs) were found in the sequences, and the translated sequences were used to search the Protein Identification Resource for comparable proteins. The ORFs were identified as: 1) an alcohol dehydrogenase, 2) an aldehyde dehydrogenase, 3) cytochrome P-450terp, 4) terpredoxin reductase, and 5) terpredoxin. The identification of both the cytochrome P-450terp and terpredoxin DNA sequence was confirmed by the presence of each of the corresponding amino acid sequences found in the purified proteins. The five ORFs were bounded on both the 5' and 3' ends by consensus factor-independent terminator sequences. A consensus promoter sequence was found immediately 5' to the first ORF. These results indicate that we have sequenced the complete terp operon. Comparison of the amino acid sequence of cytochrome P-450terp to that of all other cytochromes P-450 has shown that it is the first member of the gene family CYP108. Preliminary characterization of the chemical and physical properties and the preparation of crystals of this new cytochrome P-450, suitable for x-ray diffraction analysis, indicate that it will be useful in comparison studies with other members of this class of proteins.  相似文献   

12.
In order to study the distribution of mitochondrial cytochromes P-450 in porcine adrenal glands, the glands of anesthetized pigs were fixed in situ. Polyclonal antibodies against two cytochromes P-450, i.e., C27 side-chain cleavage enzyme and 11 beta-hydroxylase, were used to study the distribution of these enzymes in cryosections of the adrenal cortex. Ultrathin cryosections were evaluated by both protein-A/gold/silver immunocytochemistry and immunoelectron microscopy using double labeling with protein-A/colloidal-gold. At light microscopy, the two cytochrome P-450 enzymes were found to be broadly distributed in both the fasciculata and glomerulosa zones of the adrenal cortex. Quantitative immunoelectron microscopy revealed that both enzymes were localized only in mitochondria, in which they were present on the inner aspects of the inner mitochondrial membrane. Both cytochromes P-450 were demonstrable in all of the mitochondria examined, and statistical evaluation of the ratios of the two enzymes present in individual mitochondria yielded a normal distribution curve. Since no evidence was found for the preferential localization of either enzyme in a special population of mitochondria, we conclude that all mitochondria of the adrenal cortex contain both enzymes. We discuss implications of these findings with respect to the regulation of steroidogenesis.  相似文献   

13.
18- and 11beta-Hydroxylation of deoxycorticosterone and side chain cleavage of cholesterol were studied in mitochondria and submitochondrial reconstituted systems prepared from rat and bovine adrenals. A mass fragmentographic technique was used that allows determination of hydroxylation of both exogenous and endogenous cholesterol. The following results were obtained. (1) Treatment of rats with excess potassium chloride in drinking fluid increased mitochondrial cytochrome P-450 as well as 18- and 11beta-hydroxylase activity in the adrenals. Cholesterol side chain cleavage was not affected. In the presence of excess adrenodoxin and adrenodoxin reductase, cytochrome P-450 isolated from potassium chloride-treated rats had higher 18- and 11beta-hydroxylase activity per nmol than cytochrome P-450 isolated from control rats. The stimulatory effects on 18- and 11beta-hydroxylation were of similar magnitude. (2) Long-term treatment with ACTH increased cholesterol side chain cleavage in the adrenals but had no effect on 18- and 11beta-hydroxylase activity. The amount of cytochrome P-450 in the adrenals was not affected by the treatment. It was shown with isolated mitochondrial cytochrome P-450 in the presence of excess adrenodoxin and adrenodoxin reductase that the effect of ACTH was due to increase of side chain cleavage activity per nmol cytochrome P-450. Side chain cleavage of exogenous cholesterol was affected more than that of endogenous cholesterol. (3) Gel chromatography of soluble cytochrome P-450 prepared from rat and bovine adrenal mitochondria yielded chromatographic fractions having either a high 18- and 11beta-hydroxylase activity and a low cholesterol side chain cleavage activity or the reverse. The ratio between 18- and 11beta-hydroxylase activity was approximately constant, provided the origin of cytochrome P-450 was the same. (4) Addition of progesterone to incubations of deoxycorticosterone with soluble or insoluble rat adrenal cytochrome P-450 competitively inhibited 18- and 11beta-hydroxylation of deoxycorticosterone to the same degree. Addition of deoxycorticosterone competitively inhibited 11beta-hydroxylation of progesterone with the same system. Progesterone was not 18-hydroxylated by the system. From the results obtained, it is concluded that 18- and 11beta-hydroxylation have similar properties and that the binding site for deoxycorticosterone is similar or identical in the two hydroxylations. The possibility that the same specific type of cytochrome P-450 is responsible for both 18- and 11beta-hydroxylation of deoxycorticosterone is discussed.  相似文献   

14.
The interaction between cytochrome P-450 and NADPH-cytochrome c reductase during catalysis has been investigated with a reconstituted monooxygenase system composed of the two purified enzyme components and synthetic phospholipid. Steady state kinetic data are consistent with a scheme in which the formation of a binary complex between the two proteins precedes catalysis. The formation of this binary complex is described by a simple mass action equation. In agreement with this equation, the observed Vmax for benzphetamine N-demethylation was found to be directly proportional to the calculated concentration of the cytochrome P-450 . reductase complex. Furthermore, with appropriate reductase/cytochrome P-450 mole ratios, the Vmax could be shown to be linearly dependent on either the reductase or the cytochrome P-450 concentration alone. In contrast, the Km parameter is independent of the complex concentration, indicating that no change in the rate-limiting step has occurred. Thus a distinction should be made between a rate-limiting enzyme component and the rate-limiting step in this multienzyme system.  相似文献   

15.
The cytochrome P-450-dependent steroid 15 beta-hydroxylase system from Bacillus megaterium has been resolved into three components, 1) a NADPH-specific, FMN-containing flavoprotein reductase, molecular weight 55-60 000; 2) an iron-sulfur protein, molecular weight 13,000 and 3) cytochrome P-450meg, molecular weight 52,000. The cytochrome component has been purified to homogeneity, as judged by SDS-polyacrylamide gel electrophoresis and isoelectric focusing in polyacrylamide gel, and its amino acid composition has been determined. Cytochrome P-450meg has a pI of 4.9, a Stokes radius of 27 A and a sedimentation constant of 3.3 S. Electron paramagnetic resonance and optical spectra are typical of a low-spin cytochrome P-450. The fluorescence spectrum is indicative of a tryptophane residue in a relatively non-polar environment. In recombination experiments, the electron flow was shown to proceed from the reductase via the iron-sulfur protein to the cytochrome. It is also possible to exchange the different components of the mitochondrial 11 beta-hydroxylase system from bovine adrenals for corresponding components in B. megaterium. Substrate specificity studies indicate that only steroids with a 3-oxo-delta 4-configuration are hydroxylated by the B. megaterium hydroxylase system. When oxidizing agents were used, hydroxylation occurred both in positions 15 alpha and 15 beta. Further substrate specificity studies have shown that aniline and imipramine can function as substrates for the bacterial system.  相似文献   

16.
Crosslinking of protein molecules with bifunctional reagents and subsequent electrophoresis of the modified proteins revealed the presence of cytochrome P-450 LM 2 oligomers in proteoliposome membranes obtained in different ways and differing in their phospholipid composition. Data from a comparative analysis of cytochrome P-450 oligomeric structures in solution and in membrane are suggestive of the hexameric organization of cytochrome P-450 LM 2 within proteoliposome membranes.  相似文献   

17.
The binding of the amino steroid, 22-amino-23,24-bisnor-5-cholen-3 beta-ol (22-ABC), to rabbit liver cytochrome P-450 3c was studied using purified P-450 3c and liver microsomes prepared from rifampicin-treated B/J rabbits. 22-ABC binds to purified cytochrome P-450 3c producing a type II spectral change reflecting the coordination of the amine with the heme iron of the protein. In the absence of allosteric effectors, the binding is characterized by a Ks of 5 microM. In the presence of alpha-naphthoflavone or progesterone, the Ks decreases to 0.8 microM, indicating that these two compounds serve as positive effectors of the binding of 22-ABC to cytochrome P-450 3c. The antibiotic rifampicin induces cytochrome P-450 3c in rabbit liver microsomes, and the benzo(a)pyrene hydroxylase, estradiol 2-hydroxylase, and progesterone 6 beta-hydroxylase activities of these microsomes are stimulated by alpha-naphthoflavone. Moreover, the progesterone 6 beta-hydroxylase activity catalyzed by these microsomes exhibits a dependence on substrate concentration that is consistent with activation of the enzyme by the substrate, progesterone. The magnitude of the type II spectral change elicited by 22-ABC for microsomes prepared from rifampicin-treated B/J rabbits is greater than that observed for microsomes from untreated rabbits. For microsomes from rifampicin-treated rabbits, the apparent binding constant for 22-ABC was decreased 5-fold in the presence of alpha-naphthoflavone. We propose that the effects of alpha-naphthoflavone and progesterone on the binding of 22-ABC to cytochrome P-450 3c mimic the effects of the two positive effectors on the metabolism of substrates by increasing the affinity of the enzyme for substrate.  相似文献   

18.
We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.  相似文献   

19.
Previously, we described two olfactory-specific cytochromes P-450: rat cytochrome P-450olf1 (IIG1), identified by cDNA cloning, and bovine cytochrome P-450olf2 (IIA), identified by peptide microsequencing of a transmembranal polypeptide (p52). Here we describe the preparation of polyclonal antisera against peptide sequences of these proteins and their use in the immunolocalization of cytochromes P-450olf1 and P-450olf2 in rat olfactory mucosa. Immunoreactivities related to both enzymes are found in the subepithelial Bowman's glands of olfactory mucosa. Practically no immunoreactivity was found in other rat tissues, including liver, lung, kidney and respiratory mucosa. In addition, double-labeling experiments demonstrated that cytochromes P-450olf1 and P-450olf2 are present in the same population of Bowman's glands. The olfactory-specific localization of cytochromes P-450olf1 and P-450olf2 is consistent with a role for these enzymes in the modification or clearance of odorants from the chemosensory tissue.  相似文献   

20.
Streptomyces griseolus ATCC 11796 contains two inducible, herbicide-metabolizing cytochromes P-450 previously designated P-450SU1 and P-450SU2 (P-450CVA1 and P-450CVB1, respectively, using nomenclature of Nebert et al. [D. W. Nebert, M. Adesnik, M. J. Coon, R. W. Estabrook, F. J. Gonzalez, F. P. Guengerich, I. C. Gunsalus, E. F. Johnson, B. Kemper, W. Levin, I. R. Phillips, R. Sato, and M. R. Waterman, DNA 6:1-11, 1987]). Using antibodies directed against cytochrome P-450SU1, its N-terminal amino acid sequence, and amino acid composition, we cloned the suaC gene encoding cytochrome P-450SU1. Similar information about the cytochrome P-450SU2 protein confirmed that a gene cloned by cross-hybridization to the suaC gene was the subC gene encoding cytochrome P-450SU2. The suaC and subC genes were expressed in Escherichia coli, DNA for both genes was sequenced, and the deduced amino acid sequences were compared with that of the well-characterized cytochrome P-450CAM from Pseudomonas putida. Both cytochromes P-450SU1 and P-450SU2 contain several regions of strong similarity with the amino acid sequence of P-450CAM, primarily in regions of the protein responsible for attachment and coordination of the heme prosthetic group.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号