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1.
Seven cytochromes P-450 (A, B, C, D, E1, E2 and F) were isolated from hepatic microsomes of phenobarbital-induced rats by a modification of the procedure of Guengerich and Martin [Arch. Biochem. Biophys. (1980) 205, 365-379]. The modification consisted of replacing DEAE-cellulose column by two DEAE-Sepharose CL-6B columns connected in tandem, changing the elution scheme and monitoring the resulting fractions by high-pressure liquid chromatography (HPLC). Cytochrome P-450 forms D, E1, E2 and F having molecular masses of 52.5 kDa, 52.5 kDa, 53.3 kDa and 53.2 kDa, respectively were resolved from the major form of cytochrome P-450 'peak B2' of Guengerich and Martin (above reference). These four cytochromes P-450 were immunologically identical by Ouchterlony double-diffusion analysis. Slight but significant differences were evident in the partial peptide digest maps of these four cytochromes P-450 and catalytic properties of these four forms, though qualitatively similar, demonstrated distinct quantitative differences. Furthermore, HPLC retention times of these four cytochrome P-450s were quite different. Cytochrome P-450 forms A, B and C were distinctly different from each other and from the forms D, E1, E2 and F in the following respects: partial peptide digest maps, catalytic activities, and HPLC retention times. The present results show that cytochromes P-450 considered homogeneous by sodium dodecyl sulfate/polyacrylamide gel electrophoresis may be heterogeneous and contain multiple forms of cytochromes P-450 with different net charges but similar molecular-masses. These studies also demonstrate the capability of HPLC in providing a simple and effective tool for monitoring the separation of cytochromes P-450 showing charge heterogeneity. 相似文献
2.
Chromatography on 1.8-diaminooctyl-Sepharose and DEAE-Sephacel resulted in 4 fractions of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced Wistar rats. All the four fractions differed in terms of their absorption maxima in the CO-reduced state, Mr and catalytic activity. Only one cytochrome fraction (cytochrome P-450 C) possessed a high activity upon benz(a)pyrene hydroxylation. All cytochrome P-450 forms were characterized by a low rate of aminopyrine N-demethylation. Antibodies against cytochrome P-450 C (P-448) (anti-P-448) were raised. Cytochromes of fractions A, B1 and B2 in the Ouchterlony reaction of double immunodiffusion did not give precipitation bands with anti-P-448. Neither of the four cytochrome P-450 forms interacted with the antibodies raised against cytochrome P-450 isolated from liver microsomes of rats induced with phenobarbital. The procedure developed is applicable to the isolation of multiple forms of cytochrome P-450 from liver microsomes of 3-methylcholanthrene-induced rats. Using rocket immunoelectrophoresis, cytochrome P-450 C possessing a high (as compared to benz(a)pyrene metabolism) activity (18 nmol/min/nmol cytochrome) and a high (60-70%) content in 3-methylcholanthrene-induced rat liver microsomes was shown to give a relatively high yield. 相似文献
3.
S Ohmori K Motohashi M Kitada Y Kanakubo T Igarashi K Ueno H Kitagawa 《Biochemical and biophysical research communications》1984,125(3):1089-1095
Untreated monkey liver cytochrome P-450 (monkey P-450) has been purified to a specific content of 14.9 n mole/mg protein. The purified preparation was apparently homogeneous and the minimum molecular weight was estimated to be 50,000 by SDS-PAGE. Absolute spectrum of the oxidized form showed peaks at 565, 535 and 417 nm. The monkey P-450 was active in the mixed function oxidation of benzphetamine, aminopyrine, ethylmorphine, aniline and 7-ethoxycoumarin in the presence of rat liver NADPH-cytochrome P-450 reductase and DLPC. Anti monkey P-450 IgG could not inhibit rat P-450s (PB P-450, MC P-448(1) and MC P-448(2] catalyzed 7-ethoxycoumarin O-deethylation activities. 相似文献
4.
We purified two forms of cytochrome P-450 which was induced in hepatic microsomes of diabetic male rates treated with streptozotocin. One of these corresponded to P-450j. The other form, designated P450 DM-2, had a minimum molecular weight 53000 and a CO-reduced absorption maximum at 452 nm. The P450 DM-2 efficiently catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was not efficient in metabolizing aminopyrine, 7-ethoxycoumarin, aniline, N-nitrosodimethylamine, or testosterone. The NH2-terminal sequence of P450 DM-2 was identical to that of P450 K-5, the major renal cytochrome P-450. Both forms gave very similar electrophoretic patterns of proteolytic digests. P450 DM-2 and P450 K-5 are closely related forms. 相似文献
5.
Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats 总被引:1,自引:0,他引:1
M Murray 《Archives of biochemistry and biophysics》1988,262(2):381-388
Potassium ferricyanide-elicited reactivation of steroid hydroxylase activities, in hepatic microsomes from SKF 525-A-induced male rats, was used as an indicator of complex formation between individual cytochrome P-450 isozymes and the SKF 525-A metabolite. Induction of male rats with SKF 525-A (50 mg/kg for three days) led to apparent increases in androst-4-ene-3,17-dione 16 beta- and 6 beta-hydroxylation to 6.7- and 3-fold of control activities. Steroid 7 alpha-hydroxylase activity was decreased to 0.8-fold of control and 16 alpha-hydroxylation was unchanged. Ferricyanide-elicited dissociation of the SKF 525-A metabolite-P-450 complex revealed an even greater induction of 16 beta- and 6 beta-hydroxylase activities (to 1.8- and 1.6-fold of activities in the absence of ferricyanide). Androst-4-ene-3,17-dione 16 alpha-hydroxylase activity increased 2-fold after ferricyanide but 7 alpha-hydroxylase activity was unaltered. An antibody directed against the male-specific cytochrome P-450 UT-A decreased androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to 13% of control in hepatic microsomes from untreated rats. In contrast, 16 alpha-hydroxylase activity in microsomes from SKF 525-A-induced rats, before and after dissociation with ferricyanide, was reduced by anti UT-A IgG to 32 and 19% of the respective uninhibited controls. Considered together, these observations strongly suggest that the phenobarbital-inducible cytochrome P-450 isozymes PB-B and PCN-E are present in an inactive complexed state in microsomes from SKF 525-A-induced rat liver. Further, the increased susceptibility of androst-4-ene-3,17-dione 16 alpha-hydroxylase activity to inhibition by an antibody to cytochrome P-450 UT-A, following ferricyanide treatment of microsomes, suggests that this male sexually differentiated enzyme is also complexed after in vivo SKF 525-A dosage. In contrast, the constitutive isozyme cytochrome P-450 UT-F, which is active in steroid 7 alpha-hydroxylation, does not appear to be complexed to any extent in microsomes from SKF 525-A-induced rats. 相似文献
6.
A A Fa?bushevich L F Guliaeva A Iu Grishanova V M Mishin V V Liakhovich 《Biokhimii?a (Moscow, Russia)》1990,55(7):1210-1215
The synthesis of pharmacologically active diazepam metabolites (oxazepam, 4-hydroxydiazepam, N-demethyldiazepam) in liver microsomes of intact and phenobarbital-, 3-methylcholanthrene- and dexamethasone-induced male and female Wistar rats as well as in a reconstituted system with isolated forms of cytochrome P-450 (P-450a, P-450b, P-450c, P-450d and P-450k according to the Ryan nomenclature) was studied. Marked sex-dependent differences in the rates of diazepam metabolism in liver microsomes of intact and induced animals were revealed. The changes in the spectrum of diazepam metabolites in liver microsomes of induced rats (as compared to control animals) were revealed. In a reconstituted system only phenobarbital-induced cytochromes P-450b and P-450k were found to be active participants of diazepam N-demethylation; none of the isoenzymes tested were shown to be involved in diazepam hydroxylation. 相似文献
7.
The major form of cytochrome P-450, P-450K-5, was purified from kidney microsomes of untreated male rats with high-performance liquid chromatography with anion-exchange and hydroxylapatite columns. The monomeric molecular weight of P-450K-5 was 52000 on SDS-polyacrylamide gel electrophoresis and the CO-reduced absorption maximum was at 452 nm. P-450K-5 catalyzed the omega- and (omega-1)-hydroxylation of lauric acid, but was inefficient in the N-demethylation of benzphetamine and the O-dealkylation of 7-ethoxycoumarine. The NH2-terminal sequence of P-450K-5 was quite different from cytochrome P-450s purified from rat hepatic microsomes. 相似文献
8.
Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons. 相似文献
9.
T Kamataki M Shimada K Maeda R Kato 《Biochemical and biophysical research communications》1985,130(3):1247-1253
Effects of hypophysectomy and treatment with testosterone or estradiol on the sex-specific forms of cytochrome P-450, P-450-male and P-450-female, were examined. The amounts of P-450-male as well as drug oxidation activities were decreased by hypophysectomy of male rats. In female rats, drug oxidation activities were increased by hypophysectomy, which was associated with the disappearance of P-450-female and the appearance of P-450-male. Treatment of hypophysectomized female rats with testosterone or estrodiol effected minor changes in the amounts of P-450-male. 相似文献
10.
Polyclonal antibodies raised against purified trout cytochromes P-450 (P-450) LM2 (anti-LM2) and LM4b (anti-LM4b) were used in Western blot analyses with digestive gland microsomes from control and beta-naphthoflavone (BNF)-treated gumboot chitons Cryptochiton stelleri. An increase and decrease in staining intensity subsequent to treatment with anti-LM4b and anti-LM2, respectively, was observed in digestive gland microsomes from BNF-treated chiton. Thus, there appears to be at least two forms of P-450 in microsomes from the digestive gland of Cryptochiton; one of which is induced by BNF and perhaps is involved in benzo(a)pyrene (BP) biotransformation, and another form which is inhibited by BNF. 相似文献
11.
12.
Five forms of cytochrome P-450 have been purified from liver microsomes of beta-naphthoflavone-pretreated rats by chromatography on DEAE-Sephadex, DEAE-cellulose, and hydroxylapatite or CM-Sepharose columns. Over 50% of the starting cytochrome P-450 content can be accounted for in these five forms after resolution on the DEAE-cellulose column, and after further purification, the combined total recovery is 30%. The five forms have the following Mr: 47,000, 50,500, 51,500, 53,500, and 56,500. The absorption maxima in reduced carbon monoxide difference spectra are 452.5, 449, 449, 447.5, and 447.5 nm, respectively. Antibody has been prepared in rabbits to each of the five forms; each antibody reacts with the antigen for which it was prepared, but not with the other four heterologous antigens. In addition, each form gives a unique peptide map pattern when partially digested with Staphylococcus aureus V-8 protease and electrophoresed in sodium dodecyl sulfate gels. Each form also shows an individual pattern of catalytic activities when tested with benzphetamine, ethylmorphine, p-nitroanisole, benzo[alpha]pyrene, and 7-ethoxycoumarin as substrates. By all criteria examined, these five forms appear to be distinct forms of cytochrome P-450. 相似文献
13.
14.
J S Prasad R R Erickson D L Crankshaw J L Holtzman 《Archives of biochemistry and biophysics》1986,248(2):639-645
In the presence of ATP hepatic microsomes sequester calcium. This sequestration is thought to be important in the modulation of free cytosolic calcium concentration. We find that on the addition of NADPH the uptake of calcium by the hepatic microsomes is inhibited 27-85%. This inhibition is reversed by the addition of 1 mM reduced glutathione (85-91% of control), incubation under a nitrogen atmosphere (112% of control), or incubation in a 80% carbon monoxide/20% oxygen atmosphere (75% of control). Superoxide dismutase had no effect on the inhibition, while catalase reversed the inhibition by 35%. The addition of 1 mM reduced glutathione at 2 and 5 min after the addition of NADPH led to uptakes of calcium which paralleled the uptake seen when the reduced glutathione was added at the beginning of the incubation. The effect of reduced glutathione showed saturation kinetics with a Km of 10 microM. Together these data suggest that cytochrome P-450 reduces the activity of the microsomal ATP-dependent calcium pump both by the production of hydrogen peroxide and by the direct oxidation of the protein thiols. The reversal of this effect by reduced glutathione appears to be enzymatically catalyzed. 相似文献
15.
Chlorobenzene is converted to mixture of o-, m-, and p-chlorophenols in perfused rat livers, and in the following cell-free hepatic preparations from rat: postmitochondrial supernatant, microsomes, and reconstituted soluble hemoprotein systems. The percentage of m-chlorophenol steadily decreases with increasing resolution of the hemoprotein-monoxygenase system. Prior treatment of rats with 3-methylcholanthrene results in a large increase in rate of formation of the ortho isomer in all hepatic systems. Prior treatment with phenobarbital results in moderate increases in rates of formation of all three chlorophenols. Formation of the three chlorophenols is inhibited to similar extents by carbon monoxide, metyrapone, and high concentrations of glutathione. SKF-525a and 7,8-benzoflavone inhibit formation of o- and p-chlorophenols to a greater extent than that of the meta isomer; with microsomal preparations NADH greatly potentiates NADPH-dependent formation of p-chlorophenol, moderately potentiates formation of m-chlorophenol and has little effect on formation of o-chlorophenol. Dihydrodiols are not significant metabolites of Chlorobenzene with the soluble hemoprotein systems. These results, in concert with changes in proportions of phenolic metabolites seen during resolution of hepatic systems from the intact cell of the perfused liver to the soluble hemoprotein, are at least consonant with the hypothesis that chlorophenol production from Chlorobenzene is catalyzed by three different enzymes, two of which form arene oxide intermediates and one of which catalyzes a direct formation of a phenol. Thus, o- and p-chlorophenols result, respectively, on isomerization of intermediate 3- and 4-chlorobenzene oxides, while formation of m-chlorophenol would appear to occur via a direct oxidative pathway. In vitro conjugation of the arene oxide with glutathione or hydration is not a significant pathway. Assay of chlorophenols and dihydrodiols of Chlorobenzene was by high-pressure liquid chromatography. 相似文献
16.
Purification and characterization of two forms of cytochrome P-450 from rat kidney cortex microsomes
M Yoshimoto E Kusunose S Yamamoto M Maekawa M Kusunose 《Biochemistry international》1986,13(5):749-755
Two forms of cytochrome P-450 (P-450), designated P-450 k-1 and P-450 k-2, have been purified about 100-fold from rat kidney cortex microsomes. P-450 k-1 and P-450 k-2 have monomeric molecular weights of 51,500 and 52,000, respectively, on sodium dodecyl sulfate(SDS)-polyacrylamide gel electrophoresis. Absolute spectra of the oxidized forms indicate that P-450 k-1 is largely in the low-spin state and partly in the high-spin state, and that P-450 k-2 is essentially all in the former. The absorption maxima in reduced carbon monoxide difference spectra are at 450.5 and 451 nm with P-450 k-1 and P-450 k-2, respectively. The two P-450s catalyze the omega- and (omega-1)-hydroxylation of fatty acids such as caprate, laurate, myristate, and palmitate, although P-450 k-1 exhibits a higher specific activity with all fatty acids tested. In addition, P-450 k-1 is capable of hydroxylating prostaglandin (PG) A1 and A2 at the omega-position, whereas P-450 k-2 has no activity toward PGs. These activities are all stimulated by addition of cytochrome b5. The two P-450s give different peptide map patterns when partially digested with Staphylococcus aureus V8 protease or papain. 相似文献
17.
Purification and characterization of two unique forms of cytochrome P-450 from rabbit nasal microsomes 总被引:3,自引:0,他引:3
Two forms of cytochrome P-450, designated P-450NMa and P-450NMb, were purified to electrophoretic homogeneity from rabbit nasal microsomes. The purified cytochromes, which contained 14-16 nmol of P-450/mg of protein, exhibited apparent monomeric molecular weights of 49,500 and 51,000, respectively. As indicated by several criteria, including the amino acid composition, absorption spectra, and peptide maps, the two nasal forms of P-450 are distinct from each other. Furthermore, as judged by the NH2-terminal amino acid sequences, they are distinct from all other P-450 cytochromes described to date. In the ferric form, P-450NMa is in the low-spin state, whereas P-450NMb is predominantly in the high-spin state. When reconstituted with NADPH-cytochrome P-450 reductase and phospholipid, P-450NMa is very active in the oxidation of ethanol as well as several nasal procarcinogens, including the N-deethylation of N-nitrosodiethylamine, the O-deethylation of phenacetin, and the N-demethylation of hexamethyl-phosphoramide. P-450NMb also metabolizes these substrates, but at lower rates. Both nasal forms are also active with testosterone, with P-450NMa oxidizing the substrate in the 17-position to give androstenedione and P-450NMb catalyzing hydroxylation in the 15 alpha-, 16 alpha-, and 19-positions. The two cytochromes represent the major portion of the total P-450 in nasal microsomes, but the corresponding forms could not be detected in hepatic microsomes. 相似文献
18.
R.F. Struck P. Kari J. Kalin J.A. Montgomery A.J. Marinello J. Love S.K. Bansal H.L. Gurtoo 《Biochemical and biophysical research communications》1984,120(2):390-396
Incubation of [3H]-sidechain-labeled and [14C]-C(4)-ring-labeled cyclophosphamide (CPA) with purified cytochrome P-450 from liver microsomes of rats treated with phenobarbital resulted in the production of a major metabolite that contained both labels, was unaffected by diazomethane, possessed high polarity, was identical in TLC and HPLC behavior to a synthetic standard, didechlorodihydroxy-CPA, and was converted to CPA and bis(2-chloroethyl)amine by thionyl choloride. These results indicate that phenobarbital-inducible cytochrome P-450 is able to dechlorinate CPA and may account, in part, for the inability of phenobarbital to enhance the therapeutic activity and toxicity of this important anticancer and immunosuppressive agent. 相似文献
19.
Tetsuya Kamataki Kaori Maeda Yasushi Yamazoe Tsuneji Nagai Ryuichi Kato 《Archives of biochemistry and biophysics》1983,225(2):758-770
One of each constitutive form of cytochrome P-450 from liver microsomes of adult male and female rats was purified essentially following the same method to an apparent homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights estimated by the electrophoresis were 52,000 and 50,000 for forms of cytochrome P-450, P-450-male, and P-450-female, purified from male and female rats, respectively. In addition, the purified preparations of P-450-male and P-450-female showed properties different from each other with respect to spectral characteristics and catalytic activities. In Ouchterlony double diffusion plates, partially purified rabbit immunoglobulin G (IgG) raised against P-450-male and P-450-female showed very weak or no cross-reactivity with P-450-female and P-450-male, respectively. From these results, P-450-male was confirmed to be a form distinct from P-450-female. The anti-P-450-male and anti-P-450-female antibodies, which had been further purified by immunoadsorption, did not form any apparent precipitation bands with liver microsomes from untreated female and male rats, respectively. Supporting this, radial immunodiffusion analysis for P-450-male and P-450-female with an agarose gel impregnated with the rabbit antibodies showed that P-450-male and P-450-female appear in liver microsomes rather specifically depending on the sex hormones. Based on these results, sex differences in drug metabolism in the rat were confirmed as explicable, at least in part, by the presence of distinct forms of cytochrome P-450 in microsomes of male and female rats. 相似文献
20.
Multiple forms of cytochrome P-450 from kidney cortex microsomes of rabbits treated with phenobarbital 总被引:1,自引:0,他引:1
Two distinct forms of cytochrome P-450 (P-450), referred to as P-450a and P-450b, were separated and purified from kidney cortex microsomes of rabbits treated with phenobarbital. P-450a had a monomeric molecular weight of 53,000, and its CO-reduced difference spectral peak was at 450 nm. It catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1), and the omega- and (omega-1)-hydroxylation of myristate, but it was inactive toward exogenous compounds tested. On the other hand, P-450b had a monomeric molecular weight of 49,000, and its CO-reduced difference spectral peak was at 451 nm. This cytochrome was not able to hydroxylate PGA1 at all. It hydroxylated myristate much more slowly than P-450a, and preferentially at the (omega-1)-position. Unlike P-450a, P-450b efficiently metabolized exogenous compounds such as benzphetamine, aminopyrine, 7-ethoxycoumarin and p-nitroanisole. It is suggested that P-450a and P-450b are specialized for the metabolism of PGA1 and exogenous compounds, respectively, in kidney cortex microsomes. 相似文献