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1.
Regulation of cytochromes P-450 21-hydroxylase (P-450C21) and P-450 17 alpha-hydroxylase/C17,20-lyase (P-450(17) alpha,lyase) activities and impairment of this regulation by Aroclor 1254 was studied in guinea-pig adrenal microsomes. In a membrane depleted system, a decrease in the normally predominant, P-450C21 activity and an increase in P-450(17) alpha,lyase activities was observed. The same deviations were observed in intact microsomes with increase in the reaction temperature (0-40 degrees C). Breaks in Arrhenius plots for activities of P-450C21 and P-450(17) alpha,lyase correlate with transition temperatures reported for the microsomal membrane. These results point to: (1) preference of a gel state membrane for catalytic expression of P-450C21 suggesting a clustered organization of this P-450 species with reductase; (2) preference of a fluid membrane for lyase activity suggesting a random collision mechanism for reduction of P-450(17) alpha,lyase. Aroclor 1254 introduced to reaction mixtures containing intact microsomes elicited basically the same changes as caused by depletion of the microsomal membrane or by increase in the incubation temperature. Lack of effect of Aroclor 1254 on P-450C21 and P-450(17) alpha,lyase activities in the membrane depleted system demonstrates that its interference with monooxygenase activities is mediated by the microsomal membrane. The similarities between altered cytochrome P-450 mediated activities in the presence of Aroclor 1254 and the deviations observed in the membrane depleted system or upon increase in the incubation temperature may suggest that this chemical exerts its impacts by influencing membrane fluidity. 相似文献
2.
S W Karr R E Reinert A E Wade 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1985,80(1):135-139
The effects of temperature acclimation at 10, 20 and 30 degrees C on the concentration and activity of the mixed function oxidase system in bluegill are as follows. Liver weight/body weight varied inversely with temperature. Significant (P less than 0.05) differences in concentration of cytochrome P-450 of hepatic microsomes were seen and varied inversely with temperature. Benzo(a)pyrene hydroxylase activity tested in vitro at incubation temperatures of 10, 20 and 30 degrees C showed significant differences in Km, but no differences in Vmax. SDS polyacrylamide gel electrophoresis revealed some quantitative differences in cytochrome P-450 isozymes between groups. 相似文献
3.
R.K. Bhatnagar S. Ahmad K.K. Kohli K.G. Mukerji T.A. Venkitasubramanian 《Biochemical and biophysical research communications》1982,104(4):1287-1292
The presence of the components of polysubstrate monooxygenase (PSMO) activity, viz., cytochrome P-450 and NADPH cytochrome P-450 reductase has been established for the first time in the microsomes of . The microsomes were able to metabolize benzphetamine. NADPH cytochrome P-450 reductase, benzphetamine metabolism and aflatoxin production was increased by the presence of phenobarbitone (PB, 2mg/ml) in the medium. These results demonstrate that induction of PSMO activity could be a prerequisite for increased production of aflatoxins, since hydroxylation of intermediates is an obligatory step in aflatoxin biosynthesis. 相似文献
4.
Xenobiotic biotransformation in the rainbow trout liver and kidney during starvation 总被引:1,自引:0,他引:1
T Andersson U Koivusaari L F?rlin 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1985,82(1):221-225
Microsomal cytochrome P-450-dependent activities in the kidney of fish starved for 6 weeks were significantly lower than in fed fish whereas these activities in the liver were only depressed after 12 weeks of starvation. Hepatic cytochrome P-450-dependent activities were depressed to varying extents after 12 weeks of starvation when different substrates were used. The content of hepatic cytochrome P-450 was not affected by starvation. Hepatic UDP-glucuronosyl transferase activities were not affected by starvation. Induction of several hepatic cytochrome P-450-dependent activities by treatment of fish with beta-naphthoflavone was not influenced by starvation. In the kidneys of fish starved for 12 weeks induced levels of cytochrome P-450-dependent benzo(a)pyrene hydroxylase activities were significantly lower than in the kidneys of fed induced fish. 相似文献
5.
Purified adrenocortical microsomal cytochromes P-45017 alpha,lyase and P-450C21 were reconstituted with and without NADPH-cytochrome P-450 reductase in phosphatidylcholine-phosphatidylethanolamine-phosphatidylserine vesicles at a lipid to P-450 ratio of 35 (w/w) by cholate dialysis procedures. Trypsinolysis revealed that a considerable part of each P-450 molecule is deeply embedded in the lipid bilayer, on the basis of the observation of no detectable digestion for P-45017 alpha,lyase and the proteolysis-resistant membrane-bound heavy fragments for P-450C21. Rotational diffusion was measured in proteoliposomes and adrenocortical microsomes by observing the decay of absorption anisotropy, r(t), after photolysis of the heme-CO complex. Analysis of r(t) was based on a "rotation-about-membrane normal" model. The absorption anisotropy decayed within 1-2 ms to a time-independent value r3. Coexistence of a mobile population with an average rotational relaxation time phi of 138-577 microseconds and immobile (phi > or = 20 ms) populations of cytochrome P-450 was observed in both phospholipid vesicles and microsomes. Different tilt angles of the heme plane from the membrane plane were determined in proteoliposomes to be either 47 degrees or 63 degrees for P-45017 alpha,lyase from [r3/r(0)]min = 0.04 and either 38 degrees or 78 degrees for P-450C21 from [r3/r(0)]min = 0.19, when these P-450s were completely mobilized by incubation with 730 mM NaCl. Very different interactions with the reductase have been observed for the two P-450s in proteoliposomes. In the presence of the reductase, the mobile population of cytochrome P-450C21 was increased significantly from 79% to 96% due to dissociation of P-450 oligomers.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
6.
Compartmentation of ATP within renal proximal tubular cells 总被引:2,自引:0,他引:2
W Pfaller W G Guder G Gstraunthaler P Kotanko I Jehart S Pürschel 《Biochimica et biophysica acta》1984,805(2):152-157
Temperature-dependent spin changes of the heme iron atom on cytochrome P-450scc were studied by optical absorption and circular dichroism measurements. The optical absorption and circular dichroism spectra of cholesterol-free cytochrome P-450scc did not change between 10 and 26 degrees C. In contrast, the absorbance at 390 nm and the ellipticity at 330 nm of cholesterol-bound cytochrome P-450scc decreased upon temperature elevation, and the absorbance at 424 nm correspondingly increased. These spectral changes were reversible in respect of temperature. The far-ultraviolet circular dichroism spectra of both cholesterol-bound and -free cytochrome P-450scc were not affected by temperature. In addition, bound cholesterol molecule is not released from the cytochrome molecule by increasing temperature. From these results, we propose that temperature modulates specific interactions between the heme protein and bound cholesterol rather than the gross secondary structural changes of the protein. 相似文献
7.
A hepatic cytochrome P-450 isolated in an electrophoretically homogeneous form from phenobarbital-treated rats, exists predominantly in the low spin configuration (82% at 20 degrees C). The addition of saturating amounts of the substrate benzphetamine to this haemoprotein shifted the spin equilibrium to the high spin form, resulting in a doubling of the spin equilibrium constant from 0.220 to 0.539 at 20 degrees C. The histidine residues of this low spin, substrate-free cytochrome P-450 were modified in a time- and concentration-dependent manner with diethylpyrocarbonate, and progressive histidine modification resulted in a decrease of both the affinity and extent of substrate interaction with the haemoprotein. Although the histidine-modified haemoprotein maintained the capacity to undergo a temperature-dependent spin transition of the haem iron in the presence of saturating amounts of substrate, this capability was substantially decreased in comparison to the unmodified cytochrome. These results indicate that a histidine residue(s) is involved in the binding of substrate to cytochrome P-450 and hence interferes with the substrate-bound spin equilibrium. Our results further imply that histidine is probably not the sixth ligand of the substrate-free ferric form of the rat liver cytochrome P-450. 相似文献
8.
The reactions of NADPH- or dithionite-dependent reduction of cytochrome P-450 were studied using a stopped flow technique. It was found that the kinetic curves for both reactions may be fitted by a sum of the two exponents. The arrhenius plots for the fast phase rate constants are linear for both reactions. On the contrary, the breaks on the corresponding plots for the slow phase rate constants are observed at 22 and 33 degrees C for cytochrome P-450 reduction by dithionite and at 31 degrees C for NADPH-dependent reduction of cytochrome P-450. The coincidence of the values of the rate constants and activation energy (56 +/- 5 kJ/mol) for the fast phase of NADPH-dependent reduction of cytochrome P-450 with values of catalytic constants and activation energy for demethylation of tertiary amines suggests that the first electron transfer process from NADPH-cytochrome P-450 reductase to cytochrome P-450 may be the rate-limiting step. A diverse character of the kinetic parameters for the two cytochrome P-450 reduction reactions is indicative of different nature of biphasity of these processes. 相似文献
9.
Sami Ahmad Kenneth E. Kirkland Gary J. Blomquist 《Archives of insect biochemistry and physiology》1987,6(2):121-140
Direct evidence is presented for the role of a cytochrome P-450 monooxygenase (called mixed-function oxidase, or polysubstrate mono-oxygenase, PSMO) in the metabolism of the sex pheromone (Z)-9-tricosene to its corresponding epoxide and ketone in the housefly. A secondary alcohol, most likely an intermediate in the conversion of the alkene to the ketone, was also tentatively identified. The results of in vivo and in vitro experiments showed that the PSMO inhibitors, piperonyl butoxide (PB) and carbon monoxide, markedly inhibited the formation of epoxide and ketone from (9,10-3H) (Z)-9-tricosene. An examination of the relative rates of (Z)-9-tricosene metabolism showed that males exhibited a higher rate of metabolism than females with the antennae of males showing the highest activity of any tissue/organ examined. The major product from all tissues/organs was the epoxide. Data from experiments with subcellular fractions showed that the microsomal fraction had the majority of enzyme activity, which was strongly inhibited by PB and CO and required NADPH and O2 for activity. A carbon monoxide difference spectrum with reduced cytochrome showed maximal absorbance at 450 nm and allowed quantification of the cytochrome P-450 in the microsomal fraction of 0.410-nmol cytochrome P-450 mg?1 protein. Interaction of (Z)-9-tricosene with the cytochrome P-450 resulted in a type I spectrum, indicating that the pheromone binds to a hydrophobic site adjacent to the heme moiety of the oxidized cytochrome P-450. 相似文献
10.
A convenient assay for mephenytoin 4-hydroxylase activity of human liver microsomal cytochrome P-450
A simple and rapid method for the determination of (S)-mephenytoin 4-hydroxylase activity by human liver microsomal cytochrome P-450 has been developed. [Methyl-14C] mephenytoin was synthesized by alkylation of S-nirvanol with 14CH3I and used as a substrate. After incubation of [methyl-14C]mephenytoin with human liver microsomes or a reconstituted monooxygenase system containing partially purified human liver cytochrome P-450, the 4-hydroxylated metabolite of mephenytoin was separated by thin-layer chromatography and quantified. The formation of the metabolite depended on the incubation time, substrate concentration, and cytochrome P-450 concentration and was found to be optimal at pH 7.4. The Km and Vmax rates obtained with a human liver microsomal preparation were 0.1 mM and 0.23 nmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450, respectively. The hydroxylation activity showed absolute requirements for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH in a reconstituted monooxygenase system. Activities varied from 5.6 to 156 pmol 4-hydroxymephenytoin formed/min/nmol cytochrome P-450 in 11 human liver microsomal preparations. The basic system utilized for the analysis of mephenytoin 4-hydroxylation can also be applied to the estimation of other enzyme activities in which phenol formation occurs. 相似文献
11.
Y Miura 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1985,82(1):63-67
The content of cytochrome P-450 and the specific activity of laurate hydroxylation have been measured at monthly intervals through the year in liver microsomes of males and females of Japanese bullfrog, Rana catesbeiana. The specific activity, based on microsomal protein or cytochrome P-450 of both sexes was higher in spring (April-May) than in autumn (October-November) and the difference was statistically significant. However, in males the content of the cytochrome P-450 in the spring was almost the same level as in the autumn and it was rather lower in the spring than in the autumn in females. These suggest that the cytochrome P-450 species catalyzing laurate hydroxylation increased specifically in the spring. The specific activity (based on cytochrome P-450) in males was significantly higher than that in females in spring (April-May) or autumn (October-November). 相似文献
12.
《Mutation Research Letters》1993,301(2):113-119
The ability of the mussel postmitochondrial fraction (S9) to activate benzo[a]pyrene (BaP) and 2-aminoanthracene (2AA) to mutagenic metabolites towards Salmonella typhimurium strain TA98 was tested. The mechanisms involved in this activation were investigated and mussel cytochrome P-450-dependent monooxygenases and its NADPH cytochrome c reductase were found to contribute to the activation of BaP. This activation was improved by treating the mussel with 4,5,4′,5′-tetrachlorobiphenyl (TCB) (a 3-methylcholanthrene-type inducer of cytochrome P-450-dependent monooxygenase in marine fish) and was inhibited by α-naphthoflavone (ANF), a cytochrome P-450 inhibitor. However, both BaP activation and cytchrome P-450-related metabolic activities are much weaker in mussels than in vertebrates. Mussel S9 activates aromatic amines more effectively than BaP. Pretreatment of mussels with TCB or addition of ANF in the incubation medium has no effect on 2AA activation. As suggested by Kurelec (1985), aromatic amine metabolism may be supported by a flavoprotein mixed-function amine oxidase which is NADPH-dependent. 相似文献
13.
G B Vasilinin V G Lazarevich E N Korneva A I Archakov 《Biokhimii?a (Moscow, Russia)》1988,53(2):341-346
The steady-state levels of aerobic and anaerobic reduction of cytochrome b5 by ascorbic acid and the initial rates of cytochrome b5 reduction in the presence of ascorbic acid and of anaerobic cytochrome P-450 reduction in the presence of NADH were used to calculate the rate constants for cytochrome b5 oxidation. The rate constant for cytochrome b5 autooxidation in the membrane is equal to that for isolated cytochrome b5, i. e., 5 X 10(-3) s-1 (37 degrees C). The rate constant for the second cytochrome b5 oxidation reaction in the membrane, i. e., electron transfer to cytochrome P-450, is equal to 140 X 10(-3) s-1 (37 degrees C). 相似文献
14.
Studies have been performed to test the hypothesis that cytochrome P-450 from testicular microsomes consists of a single protein with two enzymatic activities (17 alpha-hydroxylase and C17,20-lyase). Three lines of evidence to support the hypothesis were obtained. (1) The enzyme appears to be homogeneous by immunochemical criteria with anti-P-450 IgG (line of identity on immunodiffusion and a single band on immunoelectrophoresis), by demonstration of a single NH2-terminal amino acid (methionine) and the finding of 16 single amino acids at the NH2 terminus. (2) Optima for pH and temperature are the same for both enzymatic activities (pH 7.25 and 37 degrees C), and temperatures between 30 and 44 degrees C decreased both activities in such a way that the ratio of hydroxylase to lyase was the same at all temperatures tested. (3) A variety of inhibitors affect both activities to the same extent: Ki values for two competitive inhibitors (SU 8000, 0.04 microM; SU 10603, 0.3 microM) are the same for hydroxylase and lyase; partition coefficients for inhibition by carbon monoxide are similar for hydroxylase and lyase (20 +/- 2 and 27 +/- 3); anti-P-450 (serum and IgG) causes inhibition of both activities to the same extent, and the same is true of a variety of less specific inhibitors. It is concluded that a single heme protein (cytochrome P-450) from microsomes of neonatal pig testis catalyzes two reactions (hydroxylase and lyase) which are sequential steps in the synthesis of androgens by the testis leading to conversion of C21 precursors to C19 steroid hormones. 相似文献
15.
G T Ankley R E Reinert A E Wade R A White 《Comp. Biochem. Physiol. C, Comp. Pharmacol. Toxicol.》1985,81(1):125-129
Bluegill (Lepomis macrochirus R.) were acclimated to 12, 22 or 32 degrees C for 5 or 14 days. Liver weight to body weight ratio and the rate of metabolism of benzo[alpha]pyrene by liver microsomes varied inversely with the acclimation temperature of the fish. Concentration of microsomal cytochrome P-450, as determined by CO-difference binding spectra, was not significantly affected by acclimation temperature. There were no qualitative or quantitative differences in the electrophoretic patterns of proteins with molecular weights similar to those reported for cytochrome P-450. There were no shifts in the temperature optima of the microsomal benzo[alpha]pyrene hydroxylase activity. 相似文献
16.
Purified hepatic NADPH-cytochrome P-450 reductase, which was reconstituted with dilauroylphosphatidylcholine, catalyzed a one-electron reductive denitrosation of 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)-1-nitrosourea ([14C]CCNU) to give 1-(2-[14C]-chloroethyl)-3-(cyclohexyl)urea at the expense of NADPH. Ambient oxygen or anoxic conditions did not alter the rates of [14C]CCNU denitrosation catalyzed by NADPH-cytochrome P-450 reductase with NADPH. Electron equivalents for reduction could be supplied by NADPH or sodium dithionite. However, the turnover number with NADPH was slightly greater than with sodium dithionite. Enzymatic denitrosation with sodium dithionite or NADPH was observed in anaerobic incubation mixtures which contained NADPH-cytochrome P-450 reductase with or without cytochrome P-450 purified from livers of phenobarbital (PB)-treated rats; PB cytochrome P-450 alone did not support catalysis. PB cytochrome P-450 stimulated reductase activity at molar concentrations approximately equal to or less than NADPH-cytochrome P-450 reductase concentration, but PB cytochrome P-450 concentrations greater than NADPH-cytochrome P-450 reductase inhibited catalytic denitrosation. Cytochrome c, FMN, and riboflavin demonstrated different degrees of stimulation of NADPH-cytochrome P-450 reductase-dependent denitrosation. Of the flavins tested, FMN demonstrated greater stimulation than riboflavin and FAD had no observable effect. A 3-fold stimulation by FMN was not observed in the absence of NADPH-cytochrome P-450 reductase. These studies provided evidence which establish NADPH-cytochrome P-450 reductase rather than PB cytochrome P-450 as the enzyme in the hepatic endoplasmic reticulum responsible for CCNU reductive metabolism. 相似文献
17.
Rat liver microsomes were immobilized by entrapment in a chemically crosslinked synthetic gel obtained by crosslinking prepolymerized polyacrylamide-hydrazide with glyoxal. Approximately 88% of the microsomal fraction was entrapped in the gel. The specific rate of O-demethylation of p-nitroanisole was used to assay the microsomal cytochrome P-450 activity of the immobilized microsomal preparations. The gel entrapped microsomes showed monooxygenase activity at 37 degrees C of Vmax = 2.3 nmol p-nitrophenol/min per nmol cytochrome P-450, similar to that of microsomes in suspension. The Km value for the p-nitroanisole-immobilized microsomal cytochrome P-450 system (1.2 X 10(-5) M) was rather close to that of microsomes in suspension (0.8 X 10(-5) M). Under the experimental conditions used the pH activity curve of the immobilized preparation was shifted towards more alkaline values by approx. 0.5 pH unit in comparison with microsomes in suspension. The rate of cytochrome c reduction by the immobilized microsomal system (11.7 nmol/min per mg protein) at 25 degrees C was considerably lower than that of the control (microsomes in suspension, 78 nmol/min per mg protein). Enzyme activity in both preparations showed the same temperature dependence at the temperature range of 10 to 37 degrees C. The immobilized microsomal monooxygenase system could be operated continuously for several hours at 37 degrees C provided that adequate amounts of an NADPH-generating system were added periodically. Under similar conditions a control microsomal suspension lost its enzymic activity within 90 min. 相似文献
18.
19.
S S Park H Miller A V Klotz P J Kloepper-Sams J J Stegeman H V Gelboin 《Archives of biochemistry and biophysics》1986,249(2):339-350
Hybridomas were prepared from myeloma cells and spleen cells of BALB/c female mice immunized with hepatic cytochrome P-450E purified from the marine fish, Stenotomus chrysops (scup). Nine independent hybrid clones produced MAbs, either IgG1, IgG2b, or IgM, that bound to purified cytochrome P-450E in radioimmunoassay. Antibodies from one clone MAb (1-12-3), also strongly recognized rat cytochrome P-450MC-B (P-450BNF-B; P-450c). The nine antibodies inhibited reconstituted aryl hydrocarbon hydroxylase (AHH) and ethoxycoumarin O-deethylase of scup cytochrome P-450E to varying degrees, and inhibited AHH activity of beta-naphthoflavone-induced scup liver microsomes in a pattern similar to that in reconstitutions, indicating that cytochrome P-450E is identical to the AHH catalyst induced in this fish by beta-naphthoflavone. MAb 1-12-3 also inhibited the reconstituted AHH activity of the major BNF-induced rat isozyme. Conversely, MAb 1-7-1 to rat cytochrome P-450MC-B had little effect on AHH activity of scup cytochrome P-450E, and did not recognize cytochrome P-450E in radioimmunoassay nor in an immunoblot. Scup cytochrome P-450E and rat cytochrome P-450MC-B thus have at least one common epitope recognized by MAb 1-12-3, but the epitope recognized by Mab 1-7-1 is absent or recognized with low affinity in cytochrome P-450E. The various assays indicate that the nine MAbs against cytochrome P-450E are directed to different epitopes of the molecule. These MAbs should be useful in determining phylogenetic relationships of the BNF- or MC-inducible isozymes and their regulation by other environmental factors. 相似文献
20.
V M Mishin E L Peregoedova D V Mishina V V Liakhovich 《Biokhimii?a (Moscow, Russia)》1989,54(7):1163-1169
Cytochrome P-450d was isolated from isosafrol-induced rat liver microsomes by affinity chromatography on 1.8-diaminooctyl-Sepharose 4B and chromatography on hydroxylapatite using a linear potassium phosphate gradient (45-250 mM). The enzyme has a molecular mass of 54 kDa, CO-maximum 448 nm is characterized by a high spin state; the rate of 4-aminobiphenyl hydroxylation is 54 nmol/min/nmol of cytochrome P-450d (37 degrees C), those, of 7-ethoxyresorufin O-deethylation and benz (a) pyrene oxidation are 1 nmol/min/nmol of cytochrome P-450d (22 degrees C) and 2 nmol/min/nmol of cytochrome P-450d (37 degrees C), respectively. The properties of cytochrome P-450d were compared to those of cytochrome P-450c isolated from 3-methylcholanthrene-induced rats. The yield of these cytochromes under the conditions used (10% P-450d from isosafrol-induced microsomes and 15% P-450c from 3-methylcholanthrene-induced microsomes) was relatively high. Antibodies to cytochromes P-450d and P-450c were obtained. Using rocket immunoelectrophoresis the percentage of these hemoprotein forms in 3-methylcholanthrene-induced (P-450d-20%, P-450c-70%) and isosafrol-induced rat liver microsomes (P-450d-50%, P-450c-15%) was determined. 相似文献