Correlation of housefly sex pheromone production with ovarian development

Pheromone production in the housefly was monitored during oögenesis and in ovariectomized insects by gas-liquid chromatography (GLC) and radio-GLC. The presence of vitellogenic ovaries was required for the initiation of (Z)-9-tricosene (muscalure), (Z)-9,10-epoxytricosane and (Z)-14-tricosen-10-one synthesis. Methylalkane synthesis was enhanced by developing ovaries. Insects ovariectomized within 12 hr after emergence produced no detectable amounts of (Z)-9-tricosene, C₂₃ epoxide nor C₂₃ ketone and synthesized less methylalkanes than the controls. This effect was reversed by ovary implants. When flies were ovariectomized after oviposition, synthesis of (Z)-9-tricosene, C₂₃ epoxide and C₂₃ ketone continued. Thus, initiation of the synthesis of these C₂₃ pheromone compounds required a vitellogenic ovary, but the ovary was not required to maintain synthesis.     

Induction of polysubstrate monooxygenase and aflatoxin production by phenobarbitone in Aspergillus parasiticus NRRL 3240

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 $\text{Aspergillus parasiticus}$. 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.     

Isoenzyme-specific phosphorylation of cytochromes P-450 and other drug metabolizing enzymes

A series of fourteen cytochrome P-450 isoenzymes was treated with three different protein kinases and found to divide into isoenzymes phosphorylated by both the cyclic AMP-dependent kinase and the calcium-phospholipid-dependent kinase (P-450 PB 3a and PB 2e), by none of these kinases (P-450 PB 1b, MC 1b, UT 1, and thromboxane synthase), and by either the cyclic AMP-dependent kinase (P-450 LM 2, PB 2d, and PB 3b) or the calcium-phospholipid-dependent kinase (P-450 PB 1a, PB 2a, MC 1a, LM 3c, and LM 4). Other components of the monooxygenase system, cytochrome P-450 reductase, cytochrome b5, cytochrome b5 reductase as well as microsomal epoxide hydrolase, were poor substrates for the kinases employed. On the other hand, glutathione transferases 1-2 and 4-4, but not 3-3, were relatively good substrates for the calcium-phospholipid-dependent kinase.     

Purification of catalytically active hepatic NADPH cytochrome P450 oxidoreductase from the rhesus monkey, Macaca mulatta.

Hepatic NADPH cytochrome P450 oxidoreductase capable of supporting polysubstrate monooxygenase (PSMO) reactions was purified from microsomes obtained from phenobarbitone (PB) pretreated rhesus monkey. Two preparations of the enzyme purified by affinity and molecular exclusion chromatographic techniques demonstrated specific content of 19.5 and 37.9 nmol cytochrome c reduced/min/mg protein and subunit molecular weight of 66 and 80 kDa, respectively. Both forms supported oxidation of NADPH and reduction of cytochrome c and DCIP but only 80 kDa preparation supported PSMO reactions. The reconstituted system consisted of hepatic P450, NADPH cytochrome P450 oxidoreductase, cytochrome b5 all purified from PB pretreated rhesus monkey and dilauroyl phosphatidylcholine or microsomal lipid. Eighty kDa preparation supported the metabolism of aminopyrine and tolbutamide by hepatic P4502C and erythromycin, ethylmorphine and nifedipine by hepatic P450 3A, respectively. The turnover of these substrates increased in the presence of partially purified cytochrome b5 from the rhesus monkey. To best of our knowledge this is the first report on the purification of monkey hepatic NADPH cytochrome P450 oxidoreductase capable of supporting in vitro PSMO by different isozymes of P450.     

Hepatic mitochondrial cytochrome P-450 system. Distinctive features of cytochrome P-450 involved in the activation of aflatoxin B1 and benzo(a)pyrene

Rat liver mitoplasts containing less than 1% microsomal contamination contain cytochrome P-450 at 25% of the microsomal level and retain the capacity for monooxygenase activation of structurally different carcinogens such as aflatoxin B1 (AFB1), benzo(a)pyrene (BaP), and dimethylnitrosamine. Both phenobarbital (PB) and 3-methylcholanthrene (3-MC) induce the level of mitochondrial cytochrome P-450 by 2.0- to 2.5-fold above the level of control mitoplasts. The enzyme activities for AFB1 (3-fold) and BaP (16-fold) metabolism were selectively induced by PB and 3-MC, respectively. Furthermore, the metabolism of AFB1 and BaP by intact mitochondria was supported by Krebs cycle substrates but not by NADPH. Both PB and 3-MC administration cause a shift in the CO difference spectrum of mitoplasts (control, 448 nm; PB, 451 nm; and 3-MC, 446 nm) suggesting that they induce two different forms of mitochondrial cytochromes P-450. Mitoplasts solubilized with cholate and fractionated with polyethylene glycol exhibit only marginal monooxygenase activities. The activity, however, was restored to preparations from both PB-induced and 3-MC-induced mitochondrial enzymes (AFB1 activation, ethylmorphine, and benzphetamine deamination and BaP metabolism) by addition of purified rat liver cytochrome P-450 reductase, and beef adrenodoxin and adrenodoxin reductase. The latter proteins failed to reconstitute activity to purified microsomal cytochromes P-450b and P-450c that were fully active with P-450 reductase. Monospecific rabbit antibodies against cytochrome P-450b and P-450c inhibited both P-450 reductase and adrenodoxin-supported activities to similar extents. Anti-P-450b and anti-P-450c provided Ouchterlony precipitin bands against PB- and 3-MC induced mitoplasts, respectively. We conclude that liver mitoplasts contain cytochrome P-450 that is closely similar to the corresponding microsomal cytochrome P-450 but can be distinguished by a capacity to interact with adrenodoxin. These inducible cytochromes P-450 are of mitochondrial origin since their levels in purified mitoplasts are over 10 times greater than can arise from the highest possible microsomal contamination.     

Induction of female sex pheromone production in male houseflies by ovary implants or 20-hydroxyecdysone

Implanting ovaries or injecting 20-hydroxyecdysone into male houseflies induced sex pheromone production, including (Z)-9-tricosene (muscalure), 9,10-epoxytricosane and (Z)-14-tricosen-10-one, which normally occurs only in vitellogenic females. Control males did not produce detectable amounts of these compounds. Injection of 20-hydroxyecdysone (5 μg/insect per day) for 3 days resulted in the accumulation of 1.81 μg/insect of (Z)-9-tricosene, 0.97 μg/insect of 9,10-epoxytricosane and 0.12 μg/insect (Z)-14-tricosen-10-one. Multiple injections of 20-hydroxyecdysone at doses as low as 50 ng resulted in the accumulation of 23:1, C₂₃ epoxide and C₂₃ ketone; shifted the distribution of label within the alkenes from 27:1 to 23:1 and decreased the amount of label in the hydrocarbon fractions as alkenes. Structures of the C₂₃ alkene and epoxide produced by the males were verified by gas chromatography-mass spectrometry. Radioactivity from [1-¹⁴C] acetate was incorporated into the C₂₃ alkene, epoxide and ketone in male insects after ovaries were implanted or they were injected with 20-hydroxyecdysone. Synthesis of the C₂₃ pheromone components decreased rapidly within several days after the administration of 20-hydroxyecdysone ceased, indicating that the enzymes involved in sex pheromone production were not permanently induced by hormone treatment. Ecdysone was also effective in initianing pheromone production in males, whereas inokosterone and cholesterol were not effective. Data presented demonstrate that male houseflies possess the metabolic capability to produce the sex pheromone components, and this suggests that 20-hydroxyecdysone alters the production of cuticular hydrocarbons such that the C₂₃ sex pheromone components become major products.     

Rotation and interaction with epoxide hydrase of cytochrome P-450 in proteoliposomes

Purified rat liver cytochrome P-450MC or P-450PB was co-reconstituted with epoxide hydrase in liposomal vesicles made of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine at a lipid to protein weight ratio of 5 by the cholate dialysis procedure. Rotational diffusion of the cytochromes was measured by observing the decay of absorption anisotropy, r(t), after photolysis of the heme.CO complex by a vertically polarized laser flash. Analysis of r(t) was based on a "rotation-about-membrane-normal" model. The measurements were used to investigate interactions of cytochrome P-450MC or P-450PB with epoxide hydrase. Different rotational mobilities of the two cytochromes were observed. The amount of mobile molecules was 78% for cytochrome P-450MC and 91% for P-450PB, and the rest was immobile within the experimental time range of 1 ms. In the presence of epoxide hydrase 85% of cytochrome P-450MC and 96% of P-450PB were mobile. Cross-linking of epoxide hydrase by anti-epoxide hydrase antibodies resulted in a drastic immobilization of the cytochromes, reducing the mobile population to 49% for P-450MC and to 60% for P-450PB. The rotational relaxation times phi of the mobile populations ranged from 210 to 283 microseconds. These results imply that both cytochromes P-450MC and P-450PB transiently associate with epoxide hydrase in liposomal membranes. Further analysis of the data showed that the angle between the heme plane of P-450MC and the membrane is 48 degrees or 62 degrees, different from the value of 55 degrees reported previously for P-450PB (Gut, J., Richter, C., Cherry, R. J., Winterhalter, K. H., and Kawato, S. (1983) J. Biol. Chem. 258, 8588-8594).     

Differential stereoselectivity on metabolism of triphenylene by cytochromes P-450 in liver microsomes from 3-methylcholanthrene- and phenobarbital-treated rats

Metabolism of triphenylene by liver microsomes from control, phenobarbital(PB)-treated rats and 3-methylcholanthrene(MC)-treated rats as well as by a purified system reconstituted with cytochrome P-450c in the absence or presence of purified microsomal epoxide hydrolase was examined. Control microsomes metabolized triphenylene at a rate of 1.2 nmol/nmol of cytochrome P-450/min. Treatment of rats with PB or MC resulted in a 40% reduction and a 3-fold enhancement in the rate of metabolism, respectively. Metabolites consisted of the trans-1,2-dihydrodiol as well as 1-hydroxytriphenylene, and to a lesser extent 2-hydroxytriphenylene. The (-)-1R,2R-enantiomer of the dihydrodiol predominated (70 to 92%) under all incubation conditions. Incubation of racemic triphenylene 1,2-oxide with microsomal epoxide hydrolase produced dihydrodiol which was highly enriched (80%) in the (-)-1R,2R-enantiomer. Experiments with 18O-enriched water showed that attack of water was exclusively at the allylic 2-position of the arene oxide, indicating that the 1R,2S-enantiomer of the oxide was preferentially hydrated by epoxide hydrolase. Thiol trapping experiments indicated that liver microsomes from MC-treated rats produced almost exclusively (greater than 90%) the 1R,2S-enantiomer of triphenylene 1,2-oxide whereas liver microsomes from PB-treated rats formed racemic oxide. The optically active oxide has a half-life for racemization of only approximately 20 s under the incubation conditions. This study may represent the first attempt to address stereochemical consequences of a rapidly racemizing intermediary metabolite.     

Induction of different isozymes of cytochrome P-450 and of microsomal epoxide hydrolase in rat liver by 2-acetylaminofluorene and structurally related compounds

The amounts of five different forms of cytochrome P-450 and of microsomal epoxide hydrolase were determined immunochemically in rat liver microsomes before and after treatment of the animals with 2-acetylaminofluorene and 15 structurally related compounds. The amount of cytochrome P-450c was found to be increased about 60-fold after treatment with 2-aminofluorene and 3-aminofluorene. Administration of 1-aminofluorene, 4-aminofluorene, 2-acetylaminofluorene and nitrofluorene increased this isozyme about 15-19 times. 2-Aminofluorene was found to inhibit the binding of 2,3,7,8-tetrachlorodibenzo-p-dioxin to a cytoplasmic receptor 50% at a concentration of 3.12 microM, while no such inhibition could be detected with 2-acetylaminofluorene. Induction of ethoxyresorufin O-deethylase activity was found to be highly correlated (+0.95) with the induction of cytochrome P-450c. Also correlated with the induction of this form was the amount of cytochrome P-450d (+0.84), which could be maximally increased about fourfold. Cytochromes P-450b + e were induced by 2-acetylaminofluorene, 4-acetylaminofluorene and fluorene (about tenfold), while 4-aminofluorene and 4-acetylaminofluorene were found to elevate cytochrome P-450PB/PCN-E about threefold. Microsomal epoxide hydrolase was induced by many of the compounds tested, with 2,7-diaminofluorene, 2,7-diacetylaminofluorene, 2-acetylaminofluorene and 2-(N-hydroxy)acetylaminofluorene being the most potent. No correlation of the induction of this enzyme with the induction of any isozyme of cytochrome P-450 was observed.     

Unique properties of NADPH- and NADH-dependent metabolism of p-nitroanisole catalyzed by cytochrome P-450 isozyme 2 in pulmonary and hepatic microsomal preparations from rabbits

Approximately 90% of the NADPH- and NADH-dependent O-demethylation of p-nitroanisole (PNA) in the hepatic microsomal fraction from phenobarbital (PB)-treated rabbits and in the pulmonary microsomal fraction from untreated rabbits is catalyzed by the same isozyme of cytochrome P-450. This isozyme of cytochrome P-450 catalyzes less than 60% of this reaction in the hepatic microsomal fraction from untreated rabbits. Antibodies to NADPH-cytochrome P-450 reductase inhibit NADPH-dependent metabolism of p-nitroanisole by about 90% but have no effect on NADH-dependent metabolism. Hepatic NADPH-dependent metabolism of pNA and reduction of cytochrome c are inhibited to the same extent with varying amounts of antibodies to NADPH cytochrome P-450 reductase. The same relationship between inhibition of monooxygenase and reductase activities is observed for the hepatic and pulmonary metabolism of benzphetamine and 7-ethoxycoumarin. In contrast, the relationship between inhibition of the pulmonary NADPH-dependent metabolism of pNA and reductase activity is biphasic; at 75% inhibition of reductase activity, metabolism of pNA is inhibited by less than 25%. For NADH-dependent metabolism of pNA, our results indicate that both electrons are transferred to cytochrome P-450 from cytochrome b5.     

Regulation of three forms of cytochrome P-450 and epoxide hydrolase in rat liver microsomes. Effects of age, sex, and induction

A procedure for the preparation of monospecific antibody directed against rat liver microsomal cytochrome P-45-a is described. This antibody, together with monospecific antibodies to cytochromes P-450b and P-450c, has been used to show that these three forms of cytochrome P-450 are distinct and share no common antigenic determinants. These antibodies (a) give single immunoprecipitin bands with detergent-solubilized microsomes; (b) do not cross-react with the purified heterologous antigens in Ouchterlony double diffusion analyses; (c) have no effect on catalytic activity of the heterologous antigens but completely inhibit the enzymatic activity of the homologous antigens; and (d) remove only the homologous antigen from detergent-solubilized microsomes when covalently bound to a solid support. With radial immunodiffusion assay, we have quantitated these three forms of cytochrome P-450 in liver microsomes after treatment of rats with seven different inducers of cytochrome P-450. The levels of these cytochrome P-450 isozymes vary independently and are also regulated by the age and sex of the animal. The antibodies have also been used to assess the contribution of cytochromes P-450a, P-450b, and P-450c in the metabolism of xenobiotics by rat liver microsomes. A large proportion of benzo(a)pyrene metabolism and testosterone 16 alpha-hydroxylation in microsomes from untreated rats is not catalyzed by cytochromes P-450a, P-450b, and P-450c. Epoxide hydrolase, another microsomal enzyme involved in the metabolism of xenobiotics, was also quantitated by radial immunodiffusion after prior treatment of rats with microsomal enzyme inducers. The inductions of epoxide hydrolase varies independently of the induction of cytochromes P-450a, P-450b, and P-450c.     

Cytochrome P-450 isozyme/isozyme functional interactions and NADPH-cytochrome P-450 reductase concentrations as factors in microsomal metabolism of warfarin

The basis for our previous observations [Kaminsky, L.S., Guengerich, F.P., Dannan, G.A. & Aust, S.D. (1983) Arch. Biochem. Biophys. 225, 398-404] that rates of microsomal metabolism of warfarin were markedly less than the sum of rates of the reconstituted constituent isozymes of cytochrome P-450 has been investigated. Metabolism of warfarin to 4'-, 6-, 7-, 8-, and 10-hydroxywarfarin and dehydrowarfarin by highly purified rat liver cytochrome P-450 (P-450) isozymes reconstituted with NADPH-cytochrome P-450 reductase and by hepatic microsomes from variously pretreated rats was used to probe functional consequences of P-450 isozyme/isozyme interactions and of the effect of microsomal reductase concentrations. Binary mixtures of P-450 isozymes were reconstituted and the regioselectivity and stereoselectivity were used to probe metabolism by each individual isozyme. The isozymes specifically inhibited each other to variable extents and the order of inhibitory potency was: P-450UT-F greater than P-450PB-D greater than or equal to P-450UT-A greater than or equal to P-450BNF/ISF-G greater than P-450PB/PCN-E greater than P-450PB-B greater than or equal to P-450PB-C greater than or equal to P-450BNF-B. The inhibition, possibly a consequence of aggregation, explains the low rate of microsomal metabolism relative to the metabolic potential of the component P-450 isozymes. When purified reductase was added to microsomes it appeared to bind to microsomes at different sites from endogenous reductase and it enhanced warfarin hydroxylase activity only to a minor extent, thus possibly precluding low reductase concentrations from being a major factor in the relatively low rates of microsomal metabolism. Antibody to the reductase differentially inhibited microsomal metabolism of warfarin by the various P-450 isozymes. The results suggest that the reductase and P-450 isozymes may be located differently relative to one another in the various microsomal preparations.     

Expression and function of three cytochrome P-450 isozymes in rat extrahepatic tissues

Western blots using a polyclonal and a monoclonal antibody raised against rat liver cytochrome P-450b indicate tissue-specific expression of low levels of cytochrome P-450's b and e. P-450b and P-450e were expressed very selectively in, respectively, lung and adrenal microsomes of untreated rats but neither isozyme was detected in the corresponding kidney or small intestine microsomes. The regioselectivity of microsomal metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) as well as the sensitivity to inhibition by anti P-450b/e IgG established that low levels of "b-like" P-450's are functional in lung and adrenal microsomes from uninduced rats, but not in microsomes from the kidney or small intestine. Functional P-450c was also detected at low levels in liver, lung, kidney, and adrenals of untreated rats. Among the extrahepatic tissues examined, DMBA metabolism was the highest in rat adrenal microsomes. However, only 30% of this activity was due to P-450's b, e, or c. Phenobarbital (PB) treatment of rats increased microsomal DMBA metabolism in all extrahepatic tissues examined. The selectivity of this increase for 12-methyl hydroxylation of DMBA and the near complete inhibition by anti-P-450b/e are consistent with induction of P-450e even though P-450b was preferentially induced in each of the extrahepatic tissues examined. The levels of expression of P-450b were increased by PB in all sets of adrenal, lung, and intestinal microsomes and in three out of six sets of kidney microsomes. The levels of P-450e were also increased by PB in all sets of adrenal microsomes. Following PB treatment, P-450e became immunoquantifiable (greater than 2 pmol/mg protein) in three of six sets of lung and kidney microsomes but remained below detection in all sets of intestinal microsomes. Based on the activity of purified P-450e, undetectable levels (less than 1 pmol/mg protein) could account for increased DMBA metabolism in this tissue. The high constitutive level of P-450b in the lung (approximately 40 pmol/mg), was remarkably inactive in DMBA metabolism and was only slightly increased by PB treatment (50%). In contrast, PB treatment caused a 2.5- to 10-fold increase in 12-methyl hydroxylation of DMBA that was highly sensitive to anti-P-450b/e. A protein comigrating with P-450e was well above detection (6-7 pmol/mg) in two of six preparations of lung microsomes that showed highest induction of this activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Immunochemical evidences that hexachlorobenzene induces two forms of cytochrome P-450 in the rat liver microsomes

Induction by hexachlorobenzene (HCB) of the liver microsomal system of metabolism of xenobiotics has been studied in comparison with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that HCB increases the content of cytochrome P-450 in the microsomes. Like PB, HCB induces the activities of aminopyrine- and benzphetamine-N-demethylases. At the same time HCB increases also the activities of benzpyrenehydroxylase and 7-ethoxyresorufin-O-deethylase, which are characteristic of the MC-induction. However, sodium dodecyl sulphate (SDS)-electrophoresis on polyacrylamide gel has revealed that HCB, similar to PB, induces protein with Mr = 52 000 (cytochrome P-450), but not the protein with Mr = 56 000, which is the main isoenzyme of cytochrome P-450 in MC-microsomes (P-448). Using specific antibodies to isolated cytochromes P-450 and P-448 (anti-P-450 and anti-P-448) it has been found by rocket immunoelectrophoresis that in HCB-treated microsomes 20% of the total cytochrome P-450 consist of PB-form and about 10% comprise cytochrome P-488. It has also been found that anti-P-448 totally inhibit 7-ethoxyresorufin-O-deethylase activity of HCB-microsomes while anti-P-450 was inactive. The data presented give direct proof that HCB exemplifies an individual chemical compound which is able to initiate the synthesis of both PB-form and MC-form of the cytochrome P-450.     

Purification of rat liver microsomal cytochrome P-450b without the use of nonionic detergent

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.     

The in vivo turnover of rat liver microsomal epoxide hydrolase and both the apoprotein and heme moieties of specific cytochrome P-450 isozymes

The in vivo turnover rates of liver microsomal epoxide hydrolase and both the heme and apoprotein moieties of cytochromes P-450a, P-450b + P-450e, and P-450c have been determined by following the decay in specific radioactivity from 2 to 96 h after simultaneous injections of NaH14CO3 and 3H-labeled delta-aminolevulinic acid to Aroclor 1254-treated rats. Total liver microsomal protein was characterized by an apparent biphasic exponential decay in specific radioactivity, with half-lives of 5-9 and 82 h for the fast- and slow-phase components, respectively. Most (approximately 90%) of the rapidly turning over microsomal protein fraction was immunologically distinct from membrane-associated serum protein, and thus appeared to represent integral membrane proteins. The existence of two distinct populations of cytochrome P-450a was suggested by the apparent biphasic turnover of both the heme and apoprotein moieties of the holoenzyme. The half-lives of the apoprotein were estimated to be 12 and 52 h for the fast- and slow-phase components, respectively, and 7 and 34 h for the heme moiety. The turnover of cytochromes P-450b + P-450e was identical to that of cytochrome P-450c, with half-lives of 37 and 28 h for the apoprotein and heme moieties, respectively. In all cases, the shorter half-lives of the heme component compared to the protein component were statistically significant. In contrast to the cytochrome P-450 isozymes, epoxide hydrolase (t1/2 = 132 h) turned over slower than the "average" microsomal protein (t1/2 = 82 h). The differential rates of degradation of these major integral membrane proteins during both the rapid and slow phases of total microsomal protein turnover argue against the concepts of unit membrane degradation and unidirectional membrane flow of liver endoplasmic reticulum.     

Purification of a new cytochrome P-450 from human liver microsomes

Using a classical methodology of purification consisting of three chromatographic steps (Octyl-Sepharose, DEAE-cellulose, CM-cellulose) we have purified a new cytochrome P-450 from human liver microsomes. It was called cytochrome P-450(9). It has been proven to be different from all precedingly purified human liver microsomal cytochrome P-450 isozymes by its immunological and electrophoretical properties. It does not cross-react with any rat liver cytochrome P-450 and anti-cytochrome P-450(9) does not recognize rat liver microsomes; thus this cytochrome P-450(9) is specific to humans. This cytochrome P-450 isozyme exists in low amounts in human liver microsomes and exhibits an important quantitative polymorphism. In reconstituted system, cytochrome P-450(9) is able to hydroxylate all substrates tested but is not specific of any; its exact role in xenobiotic metabolism in man remains to be elucidated.     

Involvement of FMN and phenobarbital cytochrome P-450 in stimulating a one-electron reductive denitrosation of 1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea catalyzed by NADPH-cytochrome P-450 reductase

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.