7 Alpha-hydroxylation of cholesterol by reconstituted systems from rat liver microsomes

A reconstituted system from rat liver microsomes, consisting of partially purified fractions of cytochrome P-450 and NADPH-cytochrome P-450 reductase was shown to catalyze 7α-hydroxylation of cholesterol in the presence of NADPH and a synthetic phosphatidylcholine. The rate of 7α-hydroxylation of added [4-¹⁴C] cholesterol was linear with the concentration of cytochrome P-450 and increased with the concentration of NADPH-cytochrome P-450 reductase up to a certain level and then remained constant. Omission of phosphatidylcholine resulted only in a 20% decrease in cholesterol 7α-hydroxylase activity of the system. The rate of 7α-hydroxylation was 2–3 times higher in reconstituted systems with cytochrome P-450 from cholestyramine-treated rats than in those with cytochrome P-450 from untreated rats.     

Studies on the rate-determining factor in testosterone hydroxylation by rat liver microsomal cytochrome P-450: evidence against cytochrome P-450 isozyme:isozyme interactions

The aim of the present study was to examine a recent proposal that inhibitory isozyme:isozyme interactions explain why membrane-bound isozymes of rat liver microsomal cytochrome P-450 exert only a fraction of the catalytic activity they express when purified and reconstituted with saturating amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. The different pathways of testosterone hydroxylation catalyzed by cytochromes P-450a (7 alpha-hydroxylation), P-450b (16 beta-hydroxylation), and P-450c (6 beta-hydroxylation) enabled possible inhibitory interactions between these isozymes to be investigated simultaneously with a single substrate. No loss of catalytic activity was observed when purified cytochromes P-450a, P-450b, or P-450c were reconstituted in binary or ternary mixtures under a variety of incubation conditions. When purified cytochromes P-450a, P-450b, and P-450c were reconstituted under conditions that mimicked a microsomal system (with respect to the absolute concentration of both the individual cytochrome P-450 isozyme and NADPH-cytochrome P-450 reductase), their catalytic activity was actually less (69-81%) than that of the microsomal isozymes. These results established that cytochromes P-450a, P-450b, and P-450c were not inhibited by each other, nor by any of the other isozymes in the liver microsomal preparation. Incorporation of purified NADPH-cytochrome P-450 reductase into liver microsomes from Aroclor 1254-induced rats stimulated the catalytic activity of cytochromes P-450a, P-450b, and P-450c. Similarly, purified cytochromes P-450a, P-450b, and P-450c expressed increased catalytic activity in a reconstituted system only when the ratio of NADPH-cytochrome P-450 reductase to cytochrome P-450 exceeded that normally found in liver microsomes. These results indicate that the inhibitory cytochrome P-450 isozyme:isozyme interactions described for warfarin hydroxylation were not observed when testosterone was the substrate. In addition to establishing that inhibitory interactions between different cytochrome P-450 isozymes is not a general phenomenon, the results of the present study support a simple mass action model for the interaction between membrane-bound or purified cytochrome P-450 and NADPH-cytochrome P-450 reductase during the hydroxylation of testosterone.     

N-demethylation of N-nitrosodimethylamine catalyzed by purified rat hepatic microsomal cytochrome P-450: isozyme specificity and role of cytochrome b5

Metabolism of the potent hepatocarcinogen N-nitrosodimethylamine (NDMA) was evaluated in reconstituted monooxygenase systems containing each of 11 purified rat hepatic cytochrome P-450 isozymes. The reaction has an absolute requirement for cytochrome P-450, NADPH-cytochrome P-450 reductase, and NADPH, as well as a partial dependence on dilauroylphosphatidylcholine. Of the cytochrome P-450 isozymes evaluated, only cytochrome P-450j, purified from livers of ethanol- or isoniazid-treated rats, had high catalytic activity for the N-demethylation of NDMA. At substrate concentrations of 0.5 and 5 mM, rates of NDMA metabolism to formaldehyde catalyzed by cytochrome P-450j were at least 15-fold greater than the rates obtained with any of the other purified isozymes. At the pH optimum (approximately 6.7) for the reaction, the Km,app and Vmax were 3.5 mM and 23.9 nmol/min/nmol cytochrome P-450j, respectively. With hepatic microsomes from ethanol-treated rats, which contain induced levels of cytochrome P-450j, the Km,app and Vmax were 0.35 mM and 3.9 nmol/min/nmol cytochrome P-450, respectively. Inclusion of purified cytochrome b5 in the reconstituted system containing cytochrome P-450j caused a six-fold decrease in Km,app (0.56 mM) of NDMA demethylation with little or no change in Vmax (29.9 nmol/min/nmol cytochrome P-450j). Trypsin-solubilized cytochrome b5, bovine serum albumin, or hemoglobin had no effect on the kinetic parameters of the reconstituted system, indicating a specific effect of intact cytochrome b5 on the Km,app of the reaction. These results demonstrate high isozyme specificity in the metabolism of NDMA to an ultimate carcinogen and further suggest an important role for cytochrome b5 in this biotransformation process.     

Cytochrome P450 IIIA1 (P450p) requires cytochrome b5 and phospholipid with unsaturated fatty acids.

In contrast to other P450 enzymes purified from rat liver microsomes, purified P450 IIIA1 (P450p) is catalytically inactive when reconstituted with NADPH-cytochrome P450 reductase and the synthetic lipid, dilauroylphosphatidylcholine. However, purified P450 IIIA1 catalyzes the oxidation of testosterone when reconstituted with NADPH-cytochrome P450 reductase, cytochrome b5, an extract of microsomal lipid, and detergent (Emulgen 911). The present study demonstrates that the microsomal lipid extract can be replaced with one of several naturally occurring phospholipids, but not with cholesterol, sphingosine, sphingomyelin, ceramide, cerebroside, or cardiolipin. The ratio of the testosterone metabolites formed by purified P450 IIIA1 (i.e., 2 beta-, 6 beta-, and 15 beta-hydroxytestosterone) was influenced by the type of phospholipid added to the reconstitution system. The ability to replace microsomal lipid extract with several different phospholipids suggests that the nature of the polar group (i.e., choline, serine, ethanolamine, or inositol) is not critical for P450 IIIA1 activity, which implies that P450 IIIA1 activity is highly dependent on the fatty acid component of these lipids. To test this possibility, P450 IIIA1 was reconstituted with a series of synthetic phosphatidylcholines. Those phosphatidylcholines containing saturated fatty acids were unable to support testosterone oxidation by purified P450 IIIA1, regardless of the acyl chain length (C6 to C18). In contrast, several unsaturated phosphatidylcholines supported testosterone oxidation by purified P450 IIIA1, and in this regard dioleoylphosphatidylcholine (PC(18:1)2) was as effective as microsomal lipid extract and naturally occurring phosphatidylcholine or phosphatidylserine. These results confirmed that P450 IIIA1 activity is highly dependent on the fatty acid component of phospholipids. A second series of experiments was undertaken to determine whether microsomal P450 IIIA1, like the purified enzyme, is dependent on cytochrome b5. A polyclonal antibody against purified cytochrome b5 was raised in rabbits and was purified by affinity chromatography. Anti-cytochrome b5 caused a approximately 60% inhibition of testosterone 2 beta-, 6 beta-, and 15 beta-hydroxylation by purified P450 IIIA1 and inhibited these same reactions by approximately 70% when added to liver microsomes from dexamethasone-induced female rats. Overall, these results suggest that testosterone oxidation by microsomal cytochrome P450 IIIA1 requires cytochrome b5 and phospholipid containing unsaturated fatty acids.     

18-Hydroxylation of deoxycorticosterone by reconstituted systems from rat and bovine adrenals.

18-Hydroxylation of deoxycorticosterone was studies with rat or bovine adrenal mitochondria or with reconstituted systems obtained from these fractions. The reconstituted systems consisted of a partially purified preparation of cytochrome P-450 from rat adrenals and a partially purified NADPH-cytochrome P450 reductase preparation from bovine adrenals. In some experimenta a soluble cytochrome P-450 fraction from bovine adrenals was used. Adrenodoxine and adrenodoxine reductase were shown to be the active components of the NADPH-cytochrome P-450 reductase preparation. Optimal assay conditions were determined for 18-hydroxylation by the crude mitochondrial fraction as well as by the reconstituted systems. In the presence of excess NADPH-cytochrome P-450 reductase fraction, the rate of 18-hydroxylation was linear with time and with the amount of cytochrome P-450. In incubations with intact rat adrenal mitochondria to which Ca2+ and an excess NADPH had been added, NADPH-cytochrome P-450 reductase increased the rate of 18-hydroxylation about 100%, indicating that NADPH-cytochrome P-45o reductase was to some extent rate-limiting. The rate of 18-hydroxylation of deoxycorticosterone by the reconstituted system as well as by intact mitochondrial fraction was much higher than the rat of 18-hydroxylation of corticosterone and progesterone. When the cytochrome P-450 preparation from rat adrenals in the reconstituted system was substituted for cytochrome P-450 from bovine adrenals, the rate of 18-hydroxylation decreased considerably. Under all experimental conditions, the 18-hydroxylation of deoxycorticosterone occurred with a concomitant and efficient 11beta-hydroxylation. Provided the source of cytochrome P-450 was the same, the ratio between 11beta- and 18hydroxylation was constant under all conditions and was not significantly different in the presence of metopirone, carbon monoxide, cytochrome c or different steroids. It is suggested that identical or at least very similar types of cytochrome P-450 are involved in 11beta- and 18-hydroxylation of deoxycorticosterone.     

Physicochemical properties of reduced nicotinamide adenine dinucleotide phosphate-cytochrome P-450 reductase from bovine adrenocortical microsomes.

Adrenocortical NADPH-cytochrome P-450 reductase (EC. 1.6.2.4) was purified from bovine adrenocortical microsomes by detergent solubilization and affinity chromatography. The purified cytochrome P-450 reductase was a single protein band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, being electrophoretically homogeneous and pure. The cytochrome P-450 reductase was optically a typical flavoprotein. The absorption peaks were at 274, 380 and 45 nm with shoulders at 290, 360 and 480 nm. The NADPH-cytochrome P-450 reductase was capable of reconstituting the 21-hydroxylase activity of 17 alpha-hydroxyprogesterone in the presence of cytochrome P-45021 of adrenocortical microsomes. The specific activity of the 21-hydroxylase of 17 alpha-hydroxyprogesterone in the reconstituted system using the excess concentration of the cytochrome P-450 reductase, was 15.8 nmol/min per nmol of cytochrome P-45021 at 37 degrees C. The NADPH-cytochrome P-450 reductase, like hepatic microsomal NADPH-cytochrome P-450 reductase, could directly reduce the cytochrome P-45021. The physicochemical properties of the NADPH-cytochrome P-450 reductase were investigated. Its molecular weight was estimated to be 80 000 +/- 1000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and analytical ultracentrifugation. The cytochrome P-450 reductase contained 1 mol each FAD and FMN as coenzymes. Iron, manganese, molybdenum and copper were not detected. The Km values of NADPH and NADH for the NADPH-cytochrome c reductase activity and those of cytochrome c for the activity of NADPH-cytochrome P-450 reductase were determined kinetically. They were 5.3 microM for NADPH, 1.1 mM for NADH, and 9-24 microM for cytochrome c. Chemical modification of the amino acid residues showed that a histidyl and cysteinyl residue are essential for the binding site of NADPH of NADPH-cytochrome P-450 reductase.     

Role of phospholipids in reconstituted cytochrome P450 3A form and mechanism of their activation of catalytic activity.

Cytochrome P-450 coded for by the 3A gene family requires specific conditions in a reconstituted system, if its catalytic activity is to be efficient. We investigated the mechanism of activation of the catalytic activity of cytochrome P450 3A by phospholipids. Rat P450 PB-1 (3A2), human P450NF (3A4), and rabbit P450 3c (3A6) were used. They had low activity in a reconstituted system (system I) with dilauroylphosphatidylcholine (DLPC) but had high activity with a mixture of phospholipids (DLPC, dioleoylphosphatidylcholine, and phosphatidylserine) and sodium cholate (system II). P450 3A forms are cationic (having a high content of lysine residues) and needed the anionic phospholipid phosphatidylserine to have sufficient activity. Double-reciprocal plots of the metabolic rate of cytochrome P-450 versus the concentration of NADPH-cytochrome P-450 reductase showed that cytochrome P-450 and the reductase interacted more in system II than in system I. P450 PB-1 did not absorb at 450 nm in the presence of reductase, CO, DLPC, and NADPH, although other cytochrome P-450s absorbed at around 450 nm in such a mixture. However, P450 PB-1 was reduced in the presence of the phospholipid mixture and sodium cholate instead of DLPC. These results suggested that the stimulation of catalytic activity by phospholipids involved increased interaction between cytochrome P-450 and the reductase. Studies of proteolytic digestion and chemical cross-linking in systems I and II showed that a P450 3A form needed disaggregation of cytochrome P-450 and/or the reductase, not the formation of an aggregated complex necessary for the catalytic activity of other cytochrome P-450s.     

Occurrence of cytochrome P-450 with prostaglandin omega-hydroxylase activity in rabbit placental microsomes

The microsomes of placenta and uterus from pregnant rabbits have been found to catalyze the omega-hydroxylation of PGE1, PGE2, PGF2 alpha, and PGA1 as well as the omega- and (omega-1)-hydroxylation of palmitate and myristate in the presence of NADPH. These activities were greatly inhibited by carbon monoxide, indicating the involvement of cytochrome P-450. The apparent Km for PGE1 was 2.38 microM and 2.1 microM with the placental and uterus microsomes, respectively. Cytochrome P-450 has been solubilized with 1% cholate from the placental microsomes, and partially purified by chromatography on 6-amino-n-hexyl Sepharose 4B, DEAE-Sephadex A-50 and hydroxylapatite columns. The partially purified cytochrome P-450 efficiently catalyzed the omega-hydroxylation of various prostaglandins such as PGE1, PGE2, PGF2 alpha, PGD2, and PGA1 in a reconstituted system containing NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. The reconstituted system also hydroxylated palmitate and myristate at the omega- and (omega-1)-position, but could not hydroxylate laurate. These catalytic properties resemble those of a new form of cytochrome P-450 highly purified from the lung microsomes of progesterone-treated rabbits (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. 96, 593-603). This type of cytochrome P-450, viz., cytochrome P-450 with high prostaglandin omega-hydroxylase activity may play a role in the regulation of prostaglandin levels in pregnancy.     

Reconstitution studies on the involvement of radiation-induced lipid peroxidation in damage to membrane enzymes

The effect of radiation on the drug-metabolizing enzyme system of microsomes, reconstituted with liposomes of microsomal phospholipids, NADPH-cytochrome P-450 reductase and cytochrome P-450, was examined to elucidate the role of lipid peroxidation of membranes in radiation-induced damage to membrane-bound enzymes. The reconstituted system of non-irradiated enzymes with irradiated liposomes showed a low activity of hexobarbital hydroxylation, whereas irradiated enzymes combined with non-irradiated liposomes exhibited an activity equal to that of unirradiated controls. Irradiation of liposomes caused a decrease in cytochrome P-450 content by destruction of the haem of cytochrome P-450 and also inhibited the binding capacity of cytochrome P-450 for hexobarbital. The relationship between radiation-induced lipid peroxidation and membrane-bound enzymes is discussed.     

Rat liver cytochrome P-450b, P-420b, and P-420c are degraded to biliverdin by heme oxygenase

In this report we provide data, for the first time, demonstrating the conversion of the heme moiety of certain cytochrome P-450 and P-420 preparations, to biliverdin, catalyzed by heme oxygenase. We have used purified preparations of cytochromes P-450c, P-450b, P-450/P-420c, or P-450/P-420b as substrates in a heme oxygenase assay system reconstituted with heme oxygenase isoforms, HO-2 or HO-1, NADPH-cytochrome c (P-450) reductase, biliverdin reductase, NADPH, and Emulgen 911. With cytochrome P-450b or P-450/P-420b preparations, a near quantitative conversion of degraded heme to bile pigments was observed. In the case of cytochrome P-450/P-420c approximately 70% of the degraded heme was accounted for as bilirubin but only cytochrome P-420c was appreciably degraded. The role of heme oxygenase in this reaction was supported by the following observations: (i) bilirubin formation was not observed when heme oxygenase was omitted from the assay system; (ii) the rate of degradation of the heme moiety was at least threefold greater with heme oxygenase and NADPH-cytochrome c (P-450) reductase than that observed with reductase alone; and (iii) the presence of Zn- or Sn-protoporphyrins (2 microM), known competitive inhibitors of heme oxygenase, resulted in 70-90% inhibition of bilirubin formation.     

Electron-transport cytochrome P-450 system is involved in the microsomal metabolism of the carcinogen chromate

The kinetics of chromate reduction by liver microsomes isolated from rats pretreated with phenobarbital or 3-methylcholanthrene with NADPH or NADH cofactor have been followed. Induction of cytochrome P-450 and NADPH-cytochrome P-450 reductase activity in microsomes by phenobarbital pretreatment caused a decrease in the apparent chromate-enzyme dissociation constant, Km, and an increase in the apparent second-order rate constant, kcat/Km, but did not affect the kcat of NADPH-mediated microsomal metabolism of chromate. Induction of cytochrome P-448 in microsomes by 3-methylcholanthrene pretreatment did not affect the kinetics of NADPH-mediated reduction of chromate by microsomes. The kinetics of NADH-mediated microsomal chromate reduction were unaffected by the drug treatments. The effects of specific enzyme inhibitors on the kinetics of microsomal chromate reduction have been determined. 2'-AMP and 3-pyridinealdehyde-NAD, inhibitors of NADPH-cytochrome P-450 reductase and NADH-cytochrome b5 reductase, inhibited the rate of microsomal reduction of chromate with NADPH and NADH. Metyrapone and carbon monoxide, specific inhibitors of cytochrome P-450, inhibited the rate of NADPH-mediated microsomal reduction of chromate, whereas high concentrations of dimethyl-sulfoxide (0.5 M) enhanced the rate. These results suggest that the electron-transport cytochrome P-450 system is involved in the reduction of chromate by microsomal systems. The NADPH and NADH cofactors supply reducing equivalents ultimately to cytochrome P-450 which functions as a reductase in chromate metabolism. The lower oxidation state(s) produced upon chromate reduction may represent the ultimate carcinogenic form(s) of chromium. These studies provide evidence for the role of cytochrome P-450 in the activation of inorganic carcinogens.     

Effects of substrates on the heat stability and on the reactivities of thiol groups of 3-phosphoglycerate kinase

The kinetics of the reduction of cytochrome P-450 LM2 mediated by NADPH-cytochrome P-450 reductase in reconstituted phospholipid vesicles was examined. An inefficient reduction of the hemoprotein in phosphatidylcholine vesicles was observed. However, by introducing negatively charged phospholipids into the membrane, the rate of reduction increased in a concomitant manner to the resulting net negative charge of the vesicles. In the presence of benzphetamine, the extent of cytochrome P-450 LM2 reduced 1 s after the addition of NADPH to the system was a linear function of the electrophoretic mobilities of the vesicles used. A similar relationship between the net negative charge of the vesicles, as measured electrophoretically, and the reduction rate was also attained in the absence of substrate. The enhanced reduction was mainly reflected in an altered phase distribution of the reduction; the extent of fast phase reduction in the absence or in the presence of added substrate was dependent upon the electrophoretic mobilities of the vesicles. A similar change in the distribution of the reduction phases was observed upon decreasing the phosphatidylcholine content of the vesicles; the fast phase reduction being more pronounced in membranes with higher relative amounts of the protein components. A decrease of the rate of O-demethylation of p-nitroanisole catalyzed by P-450 LM2 parallel to the extent of fast phase reduction was observed upon dilution of neutral phosphatidylcholine membranes with phospholipid. By contrast, no effect of lipid dilution was evident in negatively charged membranes. The results are consistent with the hypothesis that the extent of fast phase reduction is governed by the amount of complex formed between NADPH-cytochrome P-450 reductase and cytochrome P-450 in the membranes; negative membranes appear to favor the formation of such complexes, whereas similar complexes are less formed, or are not functional, in neutral membranes.     

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.     

Association of cytochrome b5 and cytochrome P-450 reductase with cytochrome P-450 in the membrane of reconstituted vesicles

A protein-protein association of cytochrome P-450 LM2 with NADPH-cytochrome P-450 reductase, with cytochrome b5, and with both proteins was demonstrated in reconstituted phospholipid vesicles by magnetic circular dichroism difference spectra. A 23% decrease in the absolute intensity of the Soret band of the magnetic CD spectrum of cytochrome P-450 was observed when it was reconstituted with reductase. A difference spectrum corresponding to a 7% decrease in absolute intensity was obtained when cytochrome b5 was incorporated into vesicles that already contained cytochrome P-450 and cytochrome P-450 reductase compared to a decrease of 13% in absolute intensity when cytochrome b5 was incorporated into vesicles that contained only cytochrome P-450. The use of the magnetic circular dichroism confirmed that protein-protein associations that have been detected by absorption spectroscopy between purified and detergent-solubilized proteins also exist in membranes. High ionic strength was shown to interrupt direct electron flow from cytochrome P-450 reductase to cytochrome P-450 but not the electron flow from reductase through cytochrome b5 to cytochrome P-450. Upon incorporation of cytochrome b5 into cytochrome P-450- and cytochrome P-450 reductase-containing vesicles, an increase of benzphetamine N-demethylation activity was observed. The magnitude of this increase was numerically identical to the residual activity of the reconstituted vesicles measured in the presence of 0.3 M KCl. It is concluded that there is a requirement for at least one charge pairing for electron transfer from reductase to cytochrome P-450. These observations are combined in a proposed mechanism of coupled reversible association reactions in the membrane.     

Comparative study of monomeric reconstituted and membrane microsomal monooxygenase systems of the rabbit liver. II. Kinetic parameters of reductase and monooxygenase reactions.

The kinetic parameters of NADPH-dependent cytochrome P450 LM2 (2B4) reduction and substrate oxidation in the monomeric reconstituted system, consisting of purified NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers, and in phenobarbital-induced rabbit liver microsomes were compared. In the absence of benzphetamine, NADPH-dependent reduction of cytochrome P450 LM2 was monophasic in the monomeric reconstituted system and biphasic in the microsomes. The presence of the substrate in the monomeric reconstituted system caused the appearance of the fast phase. In this system substrate-free cytochrome P450 LM2 was entirely low-spin, and the addition of benzphetamine shifted the spin equilibrium to a high state very weakly. No correlation between high-spin content and the proportion of the fast phase of NADPH-dependent LM2 reduction was found in the system. Vmax values for the oxidation of type I substrates (benzphetamine, dimethylaniline, aminopyrine) in the monomeric reconstituted system were higher or the same as in the microsomes, whereas Km values for the substrates and NADPH were lower in the microsomes. Maximal activity of the monomeric reconstituted system was observed at a 1:1 NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio. Measurements of benzphetamine oxidation as a function of NADPH-cytochrome P450 reductase/cytochrome P450 LM2 ratio at a constant total protein concentration allowed the Kd of the NADPH-cytochrome P450 reductase/cytochrome P450 LM2 complex to be estimated as 6.4 +/- 0.5 microM. Complex formation between the NADPH-cytochrome P450 reductase and cytochrome P450 LM2 monomers was not detected by recording the difference binding spectra of the reductase monomers with LM2 monomers or by treatment the mixture of the monomers of the proteins with the crosslinking reagent, water-soluble carbodiimide.     

Purification and characterization of ethanol-inducible human hepatic cytochrome P-450HLj

Human hepatic cytochrome P-450HLj was purified in a catalytically active state from microsomes obtained from an ethanol-intoxicated man. The electrophoretically homogeneous preparation of HLj was compared to rat P-450j and found to have a slightly different apparent molecular mass (54 vs 51.5 kDa) but highly similar immunochemical, spectral, and catalytic properties. Purified HLj exhibited high activity toward N-nitro-sodimethylamine (NDMA) and aniline metabolism, low but measurable activity toward benzphetamine and 7-ethoxycoumarin, and no detectable activity toward benzo[a]pyrene, testosterone, and progesterone. Antibody against rat P-450j reacted with HLj in immunoblot analyses and, when added directly to HLj before reconstitution with NADPH-cytochrome P-450 reductase and lipid, the antibody inhibited (96%) NDMA metabolism by HLj almost completely. However, if HLj was reconstituted with the other components before the addition of the anti-P-450j IgG, the ability of the antibody to inhibit the metabolism of NDMA was greatly diminished. This suggests that the interactions between reductase and HLj are similar to those previously observed between rat P-450j and reductase, and appear to prevent the complete access of anti-P-450j. The addition of cytochrome b5 to reconstitution systems containing HLj resulted in a small increase in the Vmax from NDMA demethylation accompanied by a decrease in Km,app (1.3 to 0.3 mM) as has been observed in reconstitution systems with rat P-450j. Therefore, in reconstituted systems, cytochrome b5 appears to play an important role in the biotransformations mediated by HLj and P-450j. In conclusion, this study demonstrates that HLj is functionally related to ethanol-inducible rat P-450j and rabbit LM3a.     

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.     

Interaction of coumarin-hydroxylating cytochrome P-450coh from liver microsomes of mice induced by pyrazole with cytochrome b5

Cytochrome P-450coh from pyrazole-treated mice was shown to form a tight and specific complex with cytochrome b5 from mouse liver microsomes. The complex formation was found to result in type I spectral changes indicating a spin shift from the low to the high spin form. When added to a reconstituted system containing cytochrome P-450coh, NADPH-cytochrome P-450 reductase and phospholipid, cytochrome b5 stimulates hydroxylation of coumarin and O-deethylation of 7-ethoxycoumarin. The maximal stimulating effect is reached at a 1:1 stoichiometry. Mouse liver cytochrome b5 stimulates hydroxylation and deethylation by 100% and 60%, respectively. The stimulating effect of cytochrome b5 was found to result from the increase of the maximal rate of oxidation, being practically without effect on Km. Cytochrome b5 purified from rat and rabbit liver microsomes interacts with cytochrome P-450coh but fails to stimulate the oxidation reaction. At large excess, cytochrome b5 inhibits the oxidations catalyzed by cytochrome P-450coh. Immobilized cytochrome b5 either from mouse or rat and rabbit microsomes proved to be an efficient affinity matrix for cytochrome P-450coh purification.     

Purification and characterization of the NADPH-cytochrome P-450 (cytochrome c) reductase from higher-plant microsomal fraction.

NADPH-cytochrome P-450 (cytochrome c) reductase (EC 1.6.2.4) was solubilized by detergent from microsomal fraction of wounded Jerusalem-artichoke (Helianthus tuberosus L.) tubers and purified to electrophoretic homogeneity. The purification was achieved by two anion-exchange columns and by affinity chromatography on 2',5'-bisphosphoadenosine-Sepharose 4B. An Mr value of 82,000 was obtained by SDS/polyacrylamide-gel electrophoresis. The purified enzyme exhibited typical flavoprotein redox spectra and contained equimolar quantities of FAD and FMN. The purified enzyme followed Michaelis-Menten kinetics with Km values of 20 microM for NADPH and 6.3 microM for cytochrome c. In contrast, with NADH as substrate this enzyme exhibited biphasic kinetics with Km values ranging from 46 microM to 54 mM. Substrate saturation curves as a function of NADPH at fixed concentration of cytochrome c are compatible with a sequential type of substrate-addition mechanism. The enzyme was able to reconstitute cinnamate 4-hydroxylase activity when associated with partially purified tuber cytochrome P-450 and dilauroyl phosphatidylcholine in the presence of NADPH. Rabbit antibodies directed against plant NADPH-cytochrome c reductase affected only weakly NADH-sustained reduction of cytochrome c, but inhibited strongly NADPH-cytochrome c reductase and NADPH- or NADH-dependent cinnamate hydroxylase activities from Jerusalem-artichoke microsomal fraction.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.