Molecular characterization of specifically active recombinant fused enzymes consisting of CYP3A4, NADPH-cytochrome P450 oxidoreductase, and cytochrome b5

Microsomal cytochrome P450 3A4 (CYP3A4) catalyzes monooxygenase reactions toward a diverse group of exogenous and endogenous substrates and requires cytochrome b5 (b5) in the oxidation of the typical substrate testosterone. To analyze the molecular interaction among CYP3A4, NADPH-cytochrome P450 oxidoreductase (P450 reductase), and b5, we constructed several fused enzyme genes and expressed them in Saccharomyces cerevisiae. The recombinant fused enzymes CYP3A4-truncated (t)-P450 reductase-t-b5 (3RB) and CYP3A4-t-b5-t-P450 reductase (3BR) in yeast microsomes showed a higher specific activity in 6beta-hydroxylation of testosterone than did the reconstitution premixes of CYP3A4, P450 reductase, and b5. The purified fused enzymes exhibited lower Km values and substantially increased Vmax values in 6beta-hydroxylation of testosterone and oxidation of nifedipine. Moreover, the fused enzymes showed significantly higher activities in cytochrome c reduction than the reconstitution premixes. Although the affinity of 3RB toward cytochrome c was twice as high as that of 3BR, 3BR and 3RB showed nearly the same affinity toward NADPH/NADH. In addition, the heme of the CYP3A4 moiety of 3RB was reduced preferentially and more rapidly than that of 3BR, whereas the heme of the b5 moiety of 3BR was selectively reduced compared with that of 3RB. These results suggest that the conformation of the 3RB molecule was the most suitable for high activity because of appropriate ordering of the CYP3A4, P450 reductase, and b5 moieties for efficient electron flow. Thus, we believe that the b5 moiety plays an important role in the efficient transfer of the second electron in the vicinity of the CYP3A4 moiety.     

Metabolism of benzo(a)pyrene,dimethylbenzanthracene and aflatoxin B1 by camel liver microsomes

The ability of camel liver microsomes to metabolise a range of common environmental carcinogens including benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 has been investigated. The camel liver has shown the ability to metabolise benzo(a)pyrene, dimethylbenzanthracene and aflatoxin B1 to a number of metabolites. The major metabolites of benzo(a)pyrene produced by camel liver enzymes were identified as its mono-hydroxy derivatives and suggest that the metabolic detoxification pathways of carcinogen metabolism are predominant in this species. Benzo(a)pyrene metabolising activity in camel liver required NADPH and was inhibited by CO and alpha-naphthoflavone suggesting the involvement of cytochrome P450 in the metabolism of this carcinogen by camel liver. The cytochrome P450-dependent metabolism of carcinogen and other specific substrates such as ethoxyresorufin and ethoxycoumarin, by camel liver enzymes, was about 50% higher than that of rat liver enzymes. The cytochrome P450-dependent metabolism of a variety of carcinogenic and other substrates by camel liver demonstrated that there are multiple forms of cytochrome P450 enzymes involved in the metabolism of a wide array of xenobiotics and pollutants.     

Mixed function oxidases in kidney and duodenum of camel,guinea pig and rat

The activities of the drug-metabolizing enzymes, aniline 4-hydroxylase, benzphetamine N-demethylase and 7-ethoxycoumarin O-deethylase have been measured in vitro in kidneys and duodenum of camels (Camelus dromedarius), guinea pigs (Cavia porcellus) and rats (Rattus norvegicus). In these species, levels of hepatic microsomal parameters namely microsomal protein, cytochrome P(450), cytochrome b(5) and NADPH-cytochrome c reductase have also been determined. In general, camels seemed to have the lowest enzyme activity when compared to rats and guinea pigs. Rats showed the highest activity in NADPH-cytochrome c reductase, aniline 4-hydroxylase and ethoxycoumarin O-deethylase among these species. However, guinea pigs showed the highest enzyme activity in cytochrome P(450), cytochrome b(5) and benzphetamine N-demethylase.     

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.     

Cytochromes P450 (CYP) in Tropical Fishes: Catalytic Activities,Expression of Multiple CYP Proteins and High Levels of Microsomal P450 in Liver of Fishes From Bermuda

Hepatic microsomes prepared from 10 fish species from Bermuda were studied to establish features of cytochrome P450 (CYP) systems in tropical marine fish. The majority (7/10) of the species had total P450 content between 0.1 and 0.5 nmol/mg, and cytochrome b₅ content between 0.025 and 0.25 nmol/mg. Ethoxycoumarin O-deethylase (ECOD) and aminopyrine N-demethylase (APND) rates in these 7 species were 0.23–2.1 nmol/min/mg and 0.5–11 nmol/min/mg, respectively, similar to rates in many temperate fish species. In contrast to those 7 species, sergeant major (Abudefduf saxatilis) and Bermuda chub (Kyphosus sectatrix) had microsomal P450 contents near 1.7 nmol/mg, among the highest values reported in untreated fish, and had greater rates of ECOD, APND, ethoxyresorufin O-deethylase (EROD) and pentoxyresorufin O-depentylase than did most of the other species. Freshly caught individuals of all species had detectable levels of EROD and aryl hydrocarbon hydroxylase (AHH) activities. Those individuals with higher rates of EROD activity had greater content of immunodetected CYP1A protein, consistent with Ah-receptor agonists acting to induce CYP1A in many fish in Bermuda waters. Injection of tomtate and blue-striped grunt with β-naphthoflavone (BNF; 50 or 100 mg/kg) induced EROD rates by 25 to 55-fold, suggesting that environmental induction in some fish was slight compared with the capacity to respond. AHH rates were induced only 3-fold in these same fish. The basis for disparity in the degree of EROD and AHH induction is not known. Rates of APND and testosterone 6β- and 16β-hydroxylase were little changed by BNF, indicating that these are not CYP1A activities in these fish. Antibodies to phenobarbital-inducible rat CYP2B1 or to scup P450B, a putative CYP2B, detected one or more proteins in several species, suggesting that CYP2B-like proteins are highly expressed in some tropical fishes. Generally, species with greater amounts of total P450 had greater amounts of proteins related to CYP2B. These species also had appreciable amounts of CYP3A-like proteins. Thus, many fishes in Bermuda appear to have induced levels of CYP1A; some also have unusually high levels of total P450 and of CYP2B-like and CYP3A-like proteins. These species may be good models for examining the structural, functional and regulatory properties of teleost CYP and the environmental or ecological factors contributing to high levels of expression of CYP in some fishes.     

Changes in the amount of cytochrome P450s in rat hepatic microsomes with starvation

The effects of starvation on the composition of 12 different cytochrome P450s in rat hepatic microsomes were studied with a specific antibody. Changes in the metabolic activity of the microsomes were studied at the same time. P450 DM (P450j) was induced 2.5-fold by a 48-h starvation and its increase reflected the increase of metabolic activity of hepatic microsomes toward aniline, 7-ethoxycoumarin, and N-nitrosodimethylamine. P450 K-5, the major renal cytochrome P450 in untreated male rat, was also induced 2.5-fold by a 48-h starvation. P450 UT-2 (P450h) and P450 UT-5 (P450g), typical male-specific forms, decreased with starvation. P450 UT-2 had high testosterone 2 alpha- and 16 alpha-hydroxylation activities. These activities of hepatic microsomes were reduced with the decrease in P450 UT-2. P450 PB-1, testosterone 6 beta-hydroxylase, was increased time-dependently by starvation. P450 UT-4 (RLM2), a minor male-specific form, was not changed by starvation. P450 PB-2 (P450k), present in both sexes, was changed little by starvation. P450 PB-4 (P450b) and P450 PB-5 (P450e) are strongly induced in rat liver by phenobarbital in coordinate fashion. Starvation increased P450 PB-4 12-fold but reduced P450 PB-5 to 22% of the control level. P450 MC-1 (P450d) was decreased by starvation. P450 MC-5 (P450c) was barely detected in control rats and was not changed by starvation. P450 IF-3 (P450a), rich in immature rats, was increased by starvation, accompanied by an increase in testosterone 7 alpha-hydroxylation activity in the hepatic microsomes. We further investigated whether new cytochrome P450s appeared upon starvation by comparison of chromatographic profiles of cytochrome P450 from starved rats with those of cytochrome P450 from control rats using HPLC. Three new cytochrome P450s were detected in the starved rats. These cytochrome P450s were purified to homogeneity. One of them was P450 DM, judging from spectral properties, catalytic activity, and the NH2-terminal sequence. The two other forms were designated P450 3b and 4b. The minimum molecular weights of P450 3b and 4b were 53,000 and 52,000, respectively, and their CO-reduced absorption maxima were at 449 and 452 nm, respectively. P450 3b metabolized aminopyrine, N-nitrosodimethylamine, 7-ethoxycoumarin, and lauric acid, but with low activity. P450 4b was efficient in lauric acid omega- and (omega-1)-hydroxylation only. The spectral properties, catalytic activity, peptide map, and NH2-terminal sequence of P450 4b agreed with those of P450 K-5. P450 3b was a new cytochrome P450, judged by these criteria.     

Reconstitution of testosterone oxidation by purified rat cytochrome P450p (IIIA1)

Cytochrome P450p (IIIA1) has been purified from rat liver microsomes by several investigators, but in all cases the purified protein, in contrast to other P450 enzymes, has not been catalytically active when reconstituted with NADPH-cytochrome P450 reductase and dilauroylphosphatidylcholine. We now report the successful reconstitution of testosterone oxidation by cytochrome P450p, which was purified from liver microsomes from troleandomycin-treated rats. The rate of testosterone oxidation was greatest when purified cytochrome P450p (50 pmol/ml) was reconstituted with a fivefold molar excess of NADPH-cytochrome P450 reductase, an equimolar amount of cytochrome b5, 200 micrograms/ml of a chloroform/methanol extract of microsomal lipid (which could not be substituted with dilauroylphosphatidylcholine), and the nonionic detergent, Emulgen 911 (50 micrograms/ml). Testosterone oxidation by cytochrome P450p was optimal at 200 mM potassium phosphate, pH 7.25. In addition to their final concentration, the order of addition of these components was found to influence the catalytic activity of cytochrome P450p. Under these experimental conditions, purified cytochrome P450p converted testosterone to four major and four minor metabolites at an overall rate of 18 nmol/nmol P450p/min (which is comparable to the rate of testosterone oxidation catalyzed by other purified forms of rat liver cytochrome P450). The four major metabolites were 6 beta-hydroxytestosterone (51%), 2 beta-hydroxytestosterone (18%), 15 beta-hydroxytestosterone (11%) and 6-dehydrotestosterone (10%). The four minor metabolites were 18-hydroxytestosterone (3%), 1 beta-hydroxytestosterone (3%), 16 beta-hydroxytestosterone (2%), and androstenedione (2%). With the exception of 16 beta-hydroxytestosterone and androstenedione, the conversion of testosterone to each of these metabolites was inhibited greater than 85% when liver microsomes from various sources were incubated with rabbit polyclonal antibody against cytochrome P450p. This antibody, which recognized two electrophoretically distinct proteins in liver microsomes from troleandomycin-treated rats, did not inhibit testosterone oxidation by cytochromes P450a, P450b, P450h, or P450m. The catalytic turnover of microsomal cytochrome P450p was estimated from the increase in testosterone oxidation and the apparent increase in cytochrome P450 concentration following treatment of liver microsomes from troleandomycin- or erythromycin-induced rats with potassium ferricyanide (which dissociates the cytochrome P450p-inducer complex). Based on this estimate, the catalytic turnover values for purified, reconstituted cytochrome P450p were 4.2 to 4.6 times greater than the rate catalyzed by microsomal cytochrome P450p.     

The microsomal oxidation system of the liver in the aging of animals with various specific lifespans

Experiments, carried out on adult and old animals with different specific lifespan (rat, guinea pig, rabbit, and dog), revealed age-related changes in content and activity of liver microsomal oxidation enzymes (cytochrome P-450, b5, aminopyrine demethylase, aniline hydroxylase). The changes become more pronounced with age. This allows to classify these species as chronobiological ones. A negative correlation between the specific lifespan and the level of decrease in activity of aminopyridine demethylase, an integral index of electron transport rate in microsomal chain, in aged animals was established.     

Constitutive testosterone 6 beta-hydroxylase in rat liver

The cytochrome P-450 that was purified from hepatic microsomes of male rats treated with phenobarbital and designated P450 PB-1 (Funae and Imaoka (1985) Biochim. Biophys. Acta 842, 119-132) had high testosterone 6 beta-hydroxylation activity (turnover rate, 13.5 nmol of product/min/nmol of P-450) in a reconstituted system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5, and a 1:1 mixture of lecithin and phosphatidylserine in the presence of sodium cholate. In ordinary conditions in the reconstituted system with cytochrome P-450, reductase, and dilauroylphosphatidylcholine, P450 PB-1 had little 6 beta-hydroxylase activity. The catalytic activities toward testosterone of two major constitutive forms, P450 UT-2 and P450 UT-5, were not affected by cytochrome b5, phospholipid, or sodium cholate. P450 PB-1 in rat liver microsomes was assayed by immunoblotting with specific antibody to P450 PB-1. P450 PB-1 accounted for 24.4 +/- 5.6% (mean +/- SD) of the total spectrally-measured cytochrome P-450 in hepatic microsomes of untreated adult male rats, and was not found in untreated adult female rats. P450 PB-1 was induced twofold with phenobarbital in male rats. P450 PB-1 was purified from untreated male rats and identified as P450 PB-1 from phenobarbital-treated rats by its NH2-terminal sequence, peptide mapping, and immunochemistry. These results showed that P450 PB-1 is a constitutive male-specific form in rat liver. There was a good correlation (r = 0.925) between the P450 PB-1 level and testosterone 6 beta-hydroxylase activity in rat liver microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)     

10-Undecynoic acid, an inhibitor of cytochrome P450 4A1, inhibits ethanolamine-specific phospholipid base exchange reaction in rat liver microsomes.

1,12-Dodecanedioic acid, the end-product of omega-hydroxylation of lauric acid, stimulates in a concentration dependent manner, phosphatidylethanolamine synthesis via ethanolamine-specific phospholipid base exchange reaction in rat liver endoplasmic reticulum. On the other hand, administration to rats of 10-undecynoic acid, a specific inhibitor of omega-hydroxylation reaction catalyzed by cytochrome P450 4A1, inhibits the ethanolamine-specific phospholipid base exchange activity by 30%. This is accompanied by a small but significant decrease in phosphatidylethanolamine content in the endoplasmic reticulum and inhibition of cytochrome P450 4A1. On the basis of these results it can be proposed that a functional relationship between cytochrome P450 4A1 and phosphatidylethanolamine synthesis exists in rat liver. Cytochrome P450 4A1 modulates the cellular level of lauric acid, an inhibitor of phospholipid synthesis. In turn, ethanolamine-specific phospholipid base exchange reaction provides molecular species of phospholipids, containing mainly long-chain polyunsaturated fatty acid moieties, required for the optimal activity of cytochrome P450 4A1.     

Complexation of membrane-bound enzyme systems

The effect of changes in the N-terminal membrane-binding domain of cytochrome P450 forms and NADPH-cytochrome P450 reductase types on the cytochrome P450-dependent monooxygenase activities, has been examined. The nifedipine oxidase activity of two human P450 forms (CYP3A4, CYP3A4NF14) which differ only in their primary structure by ten amino acid residues in the N-terminal membrane-binding domain, yields nearly the same catalytic cycle time tau =2.65 +/- 0.15 s, due to their identical cytosolic catalytic protein structure. In contrast, the complex formation process ([P450]+[reductase] <--> [complex]) described by the dissociation constant KD, at high substrate concentration ([S]>KS) and low product concentration ([P]相似文献   

Cytochrome P450-dependent monooxygenase system mediated hydrocarbon metabolism and antioxidant enzyme responses in prawn, Macrobrachium malcolmsonii

We investigated the alteration of cytochrome P450-dependent monooxygenase enzymes and antioxidant enzymes in response to oil effluent in freshwater prawn, Macrobrachium malcolmsonii. The prawns were exposed to two sublethal (10% [0.91 ppt] and 25% [2.3 ppt] of 5-day median lethal concentration) concentrations of oil. After 30 days, treated prawns were transferred into untreated freshwater and depuration was followed for another 30 days. At 7-day intervals, hydrocarbons and detoxifying enzymes were analysed in the hepatopancreas. Accumulation of hydrocarbon in the tissues gradually increased when exposed to sublethal concentration of oil effluent associated with enhanced levels of cytochrome P450, NADPH cytochrome c reductase and cytochrome b(5). During depuration, the levels of accumulated hydrocarbons decreased due to the induction of these detoxifying enzymes. Oil derived hydrocarbon mediated oxyradical production would have occurred in M. malcolmsonii. This was confirmed by elevated levels of superoxide dismutase (SOD) and catalase (CAT). Thus, cytochrome P450-dependent monooxygenase enzymes and antioxidant enzymes in oil-exposed prawns demonstrate a well-established detoxifying mechanism in M. malcolmsonii.     

Electrostatic interaction between cytochrome P450 and NADPH-P450 reductase: comparison of mixed and fused systems consisting of rat cytochrome P450 1A1 and yeast NADPH-P450 reductase

The electrostatic interaction between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase was analyzed by using recombinant yeast microsomes containing both native enzymes or their fused enzyme. The Vmax of the 7-ethoxycoumarin O-deethylation in the recombinant microsomes containing both rat cytochrome P4501A1 and yeast NADPH-P450 reductase (the mixed system) was maximal when the ionic strength of the reaction mixture was 0.1-0.15. However, on the fused enzyme between rat cytochrome P450 1A1 and yeast NADPH-P450 reductase (the fused system), the activity was uniformly reduced with increasing ionic strength. The pH profiles of Vmax were also different between the mixed and the fused systems. Based on these results, we propose a hypothesis that cytochrome P450 and NADPH-P450 reductase have more than one binding mode. The maximal activity of the mixed system at ionic strength of 0.1-0.15 is explained by change of the binding mode. On the other hand, the fused enzyme appears to have only one binding mode due to the limited topology of cytochrome P450 and NADPH-P450 reductase domains.     

Identification of cytochrome P450a (P450IIA1) as the principal testosterone 7 alpha-hydroxylase in rat liver microsomes and its regulation by thyroid hormones

In the preceding paper, evidence was presented that rat liver microsomes contain two structurally related isozymes of cytochrome P450, namely cytochromes P450a and P450m, that can both catalyze the 7 alpha-hydroxylation of testosterone. The aim of the present study was to determine the extent to which these two P450 isozymes are responsible for the 7 alpha-hydroxylation of testosterone catalyzed by rat liver microsomes. Four monoclonal antibodies against cytochrome P450a, designated A2, A4, A5, and A7, were prepared in BALB/c mice. Monoclonal antibodies A2 (an IgM), A4 (an IgG2b), and A5 (an IgG1) were determined to be distinct immunoglobulins, whereas A7 could not be distinguished from A5. All of the antibodies were highly specific for cytochrome P450a; none cross-reacted with cytochrome P450m or with 10 other P450 isozymes purified from rat liver microsomes. Competition experiments between unlabeled and horseradish peroxidase-conjugated antibodies revealed that each of the monoclonal antibodies recognized the same epitope on cytochrome P450a. None of the monoclonal antibodies bound to denatured cytochrome P450a, suggesting that they each bound to a spatial epitope. A monospecific, polyclonal antibody against cytochrome P450a was also prepared, as described in the preceding paper. The levels of cytochrome P450a in liver microsomes were determined by single radial immunodiffusion, Western immunoblot (with polyclonal antibody), and enzyme-linked immunosorbent assay with monoclonal antibody. The levels of cytochrome P450a declined with age in male but not female rats, and were inducible up to 10-fold by treatment of rats with various xenobiotics. The levels of cytochrome P450a (but not cytochrome P450m) were also elevated (approximately 3-fold) by thyroidectomy of mature male rats. Near normal levels of cytochrome P450a were restored by treatment of athyroid rats with triiodothyronine, whereas treatment with thyroxine was less effective in this regard. These changes in the levels of cytochrome P450a were highly correlated (r = 0.995) with changes in testosterone 7 alpha-hydroxylase activity. None of the monoclonal antibodies inhibited the catalytic activity of cytochrome P450a when reconstituted with NADPH-cytochrome P450 reductase and lipid. In contrast, the polyclonal antibody not only inhibited the catalytic activity of purified cytochrome P450a, but also completely inhibited (greater than 96%) the 7 alpha-hydroxylation of testosterone catalyzed by liver microsomes from immature and mature rats of both sexes and by liver microsomes from male rats treated with a variety of cytochrome P450 inducers.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maximizing the expression of mammalian cytochrome P-450 monooxygenase activities in yeast cells

Cytochrome P-450s constitute a superfamily of mono-oxygenases which require the association with specific redox enzymes bound to the endoplasmic reticulum membrane for their activity. Conditions for the functional expression of these mammalian enzymes in yeast cells and the respective merits and limitations of currently used P-450 expression systems, are considered. The dependence of the mouse P-450 IA1 specific activity on the cytochrome expression level in yeast microsomes is studied and results demonstrate that the low amounts of endogenous NADPH-cytochrome P-450 reductase and cytochrome b5 which are naturally present, are limiting for the heterologous monooxygenase activities. The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized. New vectors allowing a high level of mammalian P-450 expression upon induction were also constructed and tested. A strategy for the construction of a co-expression system allowing maximal activity of mammalian cytochrome P-450s is discussed.     

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-hydroxylase.

The isoform of cytochrome P450 that catalyzes the 12 alpha-hydroxylation of 7 alpha-hydroxy-4-cholesten-3-one, an intermediate in the conversion of cholesterol to cholic acid, was purified to homogeneity from rabbit liver microsomes. The extent of purification in the various steps was judged by an assay involving high performance liquid chromatography. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (M(r) = 50,000). The NH2-terminal amino acid sequence is as follows: Val-Leu-Trp-Gly-Leu-Leu-Gly-Ala-Leu-Leu-Met-Val-Met-Val-Gly-, which is different from that of any other P450s so far reported. The specific content of the enzyme was 13.3 nmol of cytochrome P450/mg of protein. Upon reconstitution with NADPH-cytochrome P450 reductase and cytochrome b5, the P450 enzyme showed a high activity of 12 alpha-hydroxylation with a turnover number of 36.6 min-1 at 37 degrees C. The omission of either cytochrome P450 or NADPH-cytochrome P450 reductase resulted in complete loss of activity, and the omission of cytochrome b5 resulted in 40% loss of activity. Antibodies prepared from mouse inhibited the 12 alpha-hydroxylase activity of rabbit liver microsomes about 90% and that of the rat liver microsomes 50%. The enzyme activity was not inhibited by other antibodies raised against other forms of P450 that catalyze different monooxygenation reactions toward xenobiotics or endogenous substrates. Anti-cytochrome b5 antibody inhibited the activity 40%, suggesting the functional role of this protein, and anti-reductase inhibited the activity almost completely. The microsomal enzyme activity was markedly elevated by starvation or streptozotocin administration to the animals. However, an immunoblotting experiment showed no correlation between the enzyme activity and the amount of protein, suggesting that post-translational modification may occur.     

Phosphorylation of cytochrome P450: regulation by cytochrome b5

Rabbit liver cytochrome P450 LM2 and several forms of rat liver cytochrome P450 are phosphorylated by cAMP-dependent protein kinase (PKA) and by protein kinase C. Under aqueous assay conditions at neutral pH LM2 is phosphorylated only to a maximum extent of about 20 mol% by PKA. We show that detergents or alkaline pH greatly enhance the extent of phosphorylation of the cytochrome P450 substrates of cAMP-dependent protein kinase. In the presence of 0.05% Emulgen, PBRLM5, which appears to be the best cytochrome P450 substrate for cAMP-dependent protein kinase, incorporates phosphate up to about 84 mol% of enzyme. We reported previously (I. Jansson et al. (1987) Arch. Biochem. Biophys. 259, 441-448) that cytochrome b5 inhibits the phosphorylation of LM2 by cAMP-dependent protein kinase. In this paper, using PBRLM5, we demonstrate, by analysis of initial rates, that the inhibition of phosphorylation by cytochrome b5 is competitive, with a Ki = 0.48 microM. We also show that a number of forms of cytochrome P450 can be phosphorylated by protein kinase C, and that the phosphorylation of these forms by protein kinase C is also inhibited by cytochrome b5. These data suggest that the phosphorylation site(s) of cytochromes P450 may be located within or overlap the cytochrome b5 binding domain of the enzymes.     

Phenobarbital-induced rat liver cytochrome P-450. Purification and characterization of two closely related isozymic forms

The "major" phenobarbital (PB)-induced cytochrome P-450 species present in livers of male Sprague-Dawley rats was resolved into two catalytically active heme-protein fractions on diethylaminoethyl cellulose. The two species, P-450 PB-4 (Mr = 49,000) and P-450 PB-5 (Mr = 51,000), were purified to homogeneity, and their chromatographic, spectral, catalytic, and structural properties were compared. P-450 BP-5 eluted earlier on hydroxylapatite and exhibited a more significant cholate-induced Type I spectral shift than P-450 BP-4. Very similar substrate specificity profiles were evident when the two isozymes were reconstituted with lipid, cytochrome P-450 reductase, and cytochrome b5 for oxidative metabolism of several xenobiotics, although P-450 PB-4 exhibited a higher specific catalytic activity (greater than or equal to 5-fold) with all substrates tested. Marked differences were also observed in the sensitivities of both isozymes to several P-450 inhibitors. In addition, P-450 PB-4 was greater than or equal to 10-fold more susceptible than P-450 PB-5 to suicide inactivation by two allyl-containing compounds, allylisopropylacetamide and secobarbital, providing a possible explanation of the previously observed partial inactivation by such compounds of phenobarbital-induced P-450 activity in liver microsomes. One-dimensional peptide maps of the two isoenzymes were highly similar. Antibody raised against purified Long Evans rat liver P-450b (Thomas, P. E., Korzeniowski, D., Ryan, D., and Levin, W. (1979) Arch. Biochem. Biophys. 192, 524-532) cross-reacted with P-450 PB-4 and P-450 PB-5. NH2-terminal sequence analysis demonstrated that the first 31 residues of both PB-4 and PB-5 were identical. These sequences indicated that a highly hydrophobic terminal segment, observed previously for other P-450s as well, is followed by a cluster of basic residues, suggesting that the NH2-terminal portion of these P-450s might be involved in membrane anchoring. Although it is unclear whether P-450 PB-4 and P-450 PB-5 are separate gene products or are related by post-translational modifications, this present demonstration of closely related isozymic forms suggests the possible added complexity of microheterogeneity for this family of microsomal monooxygenases.     

Production of cytochrome P450 reductase yeast-rat hybrid proteins in Saccharomyces cerevisiae.

We present a novel strategy for increasing the level of functional mammalian cytochrome P450 (Cyt.P450) and NADPH:cytochrome P450 reductase enzymes produced in yeast. A cDNA encoding the rat P450 reductase was modified by the addition of a sequence coding for the N-terminal region of P450 reductase from Saccharomyces cerevisiae. The addition of this hydrophobic tail greatly increased the apparent stability of the reductase protein produced in S. cerevisiae, as compared to the unmodified rat P450 reductase. When the rat hybrid reductase was produced simultaneously with one of two mammalian Cyt.P450s, the rat CYP2B1 or the human CYP2A6, there was a significant increase in the specific activity of each of the Cyt.P450s. The optimization of this approach and its extrapolation to other organisms should lead to a marked improvement in our ability to study and exploit the P450 system.     

Cloning and characterization of the NADPH cytochrome P450 reductase gene (CPR) from Candida bombicola

Candida bombicola is a yeast with at least two appealing features. The species can grow on alkanes when provided as the sole carbon source, and it produces glycolipids, which have several industrial, cosmetic and pharmaceutical applications. Both metabolic processes require in their pathway the activity of cytochrome P450 monooxygenase. This enzyme needs and gets reducing equivalents from NADPH cytochrome P450 reductase (CPR). The CPR gene of Candida bombicola was isolated using degenerate PCR and genomic walking. The gene encodes an enzyme of 687 amino acids, which shows homology with known CPRs of other species. The functionality of the gene was proven by heterologous expression in Escherichia coli. The recombinant protein exhibited NADPH-dependent cytochrome c reducing activity. Cloning and characterization of this enzyme is an important step in the study of the cytochrome P450 monooxygenase system of Candida bombicola. The GenBank accession number of the sequence described in this article is EF050789.