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.     

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 effect of some biphenyl solubilizing and suspending agents on biphenyl 4-hydroxylase of rabbit liver microsomes

The effects of ethanol, acetone, dimethylsulfoxide (DMSO), polyoxyethylene sorbitan monooleate (Tween 80), polyoxyethylene sorbitan monolaurate (Tween 20), Triton X-100, and carboxymethyl cellulose (CMC) on the kinetics of biphenyl 4-hydroxylase of rabbit liver microsomes were investigated in an attempt to find a substrate-solubilizing or suspending agent (carrier) which was itself a non-effector of the mixed-function oxidase. The effects of these carriers on the activities of NADPH-cytochrome P-450 reductase, NADPH-cytochrome c reductase, and cytochrome P-450 content were also investigated.Ethanol and DMSO inhibited biphenyl 4-hydroxylase and NADPH-cytochrome P-450 reductase. Acetone inhibited the hydroxylase uncompetitively at concentrations which appeared to stimulate NADPH-cytochrome P-450 reductase. All of the detergents inhibited biphenyl 4-hydroxylase although only Triton X-100 markedly affected the reduction of cytochrome P-450. The interaction of Tween 80 with the hydroxylase gave rise to non-linear Lineweaver-Burk plots although at high concentrations of biphenyl or low concentrations of the detergent the inhibition appeared to be competitive.Biphenyl caused a 2–3-fold stimulation of NADPH-cytochrome P-450 reductase, but in the presence of Tween 80 the stimulation was absent. Since V of biphenyl 4-hydroxylase in the presence of Tween 80 was not significantly different from V in its absence it would appear that the reduction of cytochrome P-450 was not ratelimiting.Of all the carriers studied only CMC was without effect on all aspects of microsomal electron transport investigated. As far as biphenyl 4-hydroxylase is concerned, CMC appears to be the most suitable substrate carrier.     

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].     

Reconstituted liver microsomal enzyme system that hydroxylates drugs, other foreign compounds and endogenous substrates. VII. Stimulation of benzphetamine N-demethylation by lipid and detergent

The resolved liver microsomal hydroxylation system required lipid for benzphetamine N-demethylation. Certain nonionic detergents, such as Emulgen 911, Triton N-101, and Triton X-100, at appropriate concentrations could substitute for lipid. These results suggest that lipid and detergent activate the cytochrome P-450-containing hydroxylation system by a similar mechanism, probably by enhancing the interaction between cytochrome P-450 and NADPH-cytochrome c reductase.     

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Metabolism of dichlorobiphenyls by highly purified isozymes of rat liver cytochrome P-450

Hepatic mixed-function oxidase metabolism of the ubiquitous pollutant polychlorinated biphenyls (PCBs) is implicated in their toxification and detoxification. We used dichlorobiphenyls (DCBs) as models to investigate the effect of the chloro substituent sites on this metabolism experimentally and by molecular orbital calculations. Reconstituted, purified cytochrome P-450 PB-B and BNF-B, the major terminal oxidase isozymes of this system, from phenobarbital (PB)- and beta-naphthoflavone (BNF)-induced rats were used to investigate this metabolism. Both isozymes are also induced by PCBs. High-performance liquid chromatography (HPLC) was used to detect, quantify, and isolate metabolites. Metabolite structures were identified by mass spectrometry, dechlorination to identifiable hydroxybiphenyls, and HPLC retention times. All DCBs yielded 3- and 4- but no 2-monohydroxylated metabolites (3,3'-DCB also yielded a dihydroxy metabolite). Di-o-chloro-substituted DCBs were metabolized primarily by cytochrome P-450 PB-B, mono-o-chloro substituted DCBs by both isozymes approximately equivalently, and DCBs without o-chloro substituents by BNF-B primarily. Thus PB-B preferentially metabolizes noncoplanar DCBs and BNF-B coplanar DCBs. The cytochrome isozymes exhibited differing regioselectivities for DCB metabolism - PB-B hydroxylated unchlorinated phenyl rings and BNF-B chlorinated rings. Incorporation of epoxide hydrolase yielded DCB dihydrodiols, and hydroxy metabolite patterns were consistent with those calculated from ring-opened arene oxide intermediates. Thus the rates and regioselectivities of metabolism and thus possibly the toxicity and carcinogenicity of DCBs are dependent on the cytochrome P-450 isozymes induced.     

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.     

Complexation of cytochrome P-450 isozymes in hepatic microsomes from SKF 525-A-induced rats

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.     

Immobilized cytochrome P-450LM2. Dissociation and reassociation of oligomers.

Subunit interactions in the purified hexameric cytochrome P-450_LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450_LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.     

Reconstruction of a membrane monooxygenase cytochrome P 450-containing system in the liver using detergents in solution

Emulgen 913, Triton N-101 and sodium cholate were compared for their reconstituting action on the dimethylaniline N-demethylation system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The comparison showed that emulgen 913 is the most efficient detergent. The optimum molar ratio of the proteins and emulgen appeared to be equal to 1:1:600. Study on the mechanism of emulgen reconstituting action showed that this effect is due to the mixed complex formation between the cytochrome and reductase, the complexes containing five molecules of the flavoprotein and five molecules of cytochrome P-450. No formation of mixed protein aggregates or reconstitution was observed in the absence of the detergent or at its concentrations exceeding the optimum level.     

Purification and characterization of human placental aromatase cytochrome P-450

Aromatase cytochrome P-450, which catalyzes the conversion of androgens to estrogens, was purified from human placental microsomes. The enzyme was extracted with sodium cholate, fractionated by ammonium sulfate precipitation, and subjected to column chromatography in the presence of its substrate, androstenedione, and the nonionic detergent, Nonidet P-40. The preparation exhibits a single major band when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and has a specific content of 11.5 nmol of P-450/mg of protein. The purified enzyme displays spectroscopic properties typical of the ferric and ferrous forms of cytochrome P-450. Full enzymatic activity can be reconstituted with rabbit liver microsomal cytochrome P-450 reductase and Nonidet P-40. Purified aromatase cytochrome P-450 displays catalytic characteristics similar to the enzyme in intact microsomes in the aromatization of androstenedione, 19-hydroxyandrostenedione and 19-oxoandrostenedione. Testosterone and 16 alpha-hydroxytestosterone are aromatized at maximal rates similar to androstenedione, and all substrates exhibit relative affinities corresponding to those observed in microsomes. We have raised rabbit antibodies to the purified enzyme which show considerable specificity and sensitivity on immunoblots.     

Quantitative determination of zwitterionic detergents using salt-induced phase separation of Triton X-100

Zwitterionic detergents interfere with the salt-induced phase separation for nonionic detergents in a concentration-dependent manner by shifting the normal cloud point of nonionic detergents to a higher ionic strength at room temperature. This phenomenon was used to determine the concentration of the zwitterionic detergents CHAPS, CHAPSO, and sulfobetaine SB-12 in solution by titration with ammonium sulfate in the presence of Triton X-100. Among the ionic detergents tested, the method was only applicable to sodium cholate. The assay can be used to control the removal of zwitterionic detergents during the reconstitution of membrane proteins in liposomes. However, it cannot be used to determine the specific binding of zwitterionic detergents to highly diluted, pure membrane proteins because of the limited sensitivity. Neither proteins nor phospholipids interfered with this method at concentrations up to 20 mg/ml of test solution (human serum albumin) or 10 mg/ml (phospholipids), respectively. Since the assay is based on the competition between salts and nonionic detergents for water molecules, it is important to equalize the ionic strength of samples and calibration standards.     

Effect of detergents on the N-and ring-hydroxylation of 2-acetamidofluorene by hamster liver microsomal preparations.

Effects of detergents such as cholate, deoxycholate and Triton X-100 were studied on N-and ring-hydroxylation of 2-acetamidofluorene by reconstituted and unresolved microsomal systems from livers of hamsters pretreated with 3-methylcholanthrene. Triton X-100 (2.5 mg/nmol of cytochrome P-448) inhibited N-and ring-hydroxylation by wholemicrosomal preparations by 40 and 90% respectively Deoxycholate at the same concentration inhibited both hydroxylations completely, whereas cholate inhibited N-and ring-hydroxylation by 40 and 50% respectively. In reconstitution studies, the presence of Triton X-100(0.5-1.0mg/nmol of cytochrome P-448) along with unsolubilized cytochrome P-448 fraction and solubilized reductase fraction increased N-hydroxylation to an appreciable extent compared with ring-hydroxylation. Both cholate and deoxycholate at 0.5-1.0 mg concentrations had a greater stimulatory effect on ring-than on N-hydroxylation activity in such a reconstituted system.     

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

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.     

Monoclonal antibodies to liver microsomal cytochrome P-450E of the marine fish Stenotomus chrysops (scup): cross reactivity with 3-methylcholanthrene induced rat cytochrome P-450

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.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Immobilized cytochrome P-450LM2: Dissociation and reassociation of oligomers

Subunit interactions in the purified hexameric cytochrome P-450_LM2 have been studied using covalent binding of one of the 6 protomers to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (> 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450_LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.     

The participation of a second molecule of adrenodoxin in cytochrome P-450-catalyzed 11beta hydroxylation.

We have utilized 11beta-hydroxylase activity and visible absorption spectrophotometry to detect possible complex formation among adrenodoxin reductase, adrenodoxin, and cytochrome P-450(11)beta. At low ionic strength, a 1:1 complex between adrenodoxin reductase and adrenodoxin occurs but does not support maximal rates of 11beta hydroxylation; at least 1 additional molecule of adrenodoxin in excess of the 1:1 complex is required for full hydroxylase activity. Spectrophotometric titration of a mixture of adrenodoxin reductase and cytochrome P-450(11)beta with adrenodoxin indicates sequential formation of 1:1 complexes between adrenodoxin reductase and adrenodoxin and then between a second adrenodoxin and cytochrome P-450(11beta; the adrenodoxin-cytochrome P-450(11)beta complex is only detectable when the concentration of adrenodoxin exceeds that of adrenodoxin reductase.     

Characterization of a cytochrome P-450 dependent monoterpene hydroxylase from the higher plant Vinca rosea.

A monooxygenase isolated from 5-day old etiolated Vinca rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was inpendent upon NADPH (neither NADH nor combination of NADH, NADP+ and ATP served as substitutes) and O2. Geraniol hydroxylation was enhanced by dithiothreitol (monothiols were less effective) and inhibited by phospholipases, thiol reagents, metyrapone, and cytochrome c, as well as other inhibitors of cytochrome P-450 systems. Geraniol was hydroxylated at a faster rate than nerol, but the alcohols possessed similar apparent Km values. The membrane-bound hydroxylase was solubilized by treatment with sodium cholate, Renex-30, or Lubrol-WX. Cholate-treated enzyme was resolved by DEAE-cellulose chromatography and reconstitution of the hydroxylase was effected utilizing different fractions containing cytochrome P-450, a NADPH-cytochrome c reductase, and lipid.