The effects of phosphatase on the components of the cytochrome P-450-dependent microsomal monooxygenase

Incubation of rabbit liver microsomes with alkaline phosphatase resulted in a marked decrease of NADPH-dependent monooxygenase activities. This decrease was found to be correlated with the decrease of NADPH-cytochrome c reductase activity catalyzed by NADPH-cytochrome P-450 reductase. Neither the content of cytochrome P-450, as determined from its CO difference spectrum, nor the peroxide-supported demethylase activity catalyzed by cytochrome P-450 alone was affected by the phosphatase treatment. NADH-cytochrome b5 reductase and cytochrome b5 were not affected by the phosphatase either. NADPH-cytochrome P-450 reductase purified from rabbit liver microsomes lost its NADPH-dependent cytochrome c reductase activity upon incubation with phosphatase in a way similar to that of microsome-bound reductase. Flavin analysis showed that the phosphatase treatment caused a decrease of FMN with concomitant appearance of riboflavin. Alkaline phosphatase, therefore, inactivates the reductase by attacking its FMN, and the inactivation of the reductase, in turn, leads to a decrease of the microsomal monooxygenase activities.     

Effect of monooxygenase reactions catalyzed by cytochrome P-450 on the microsomal membrane

Hydroxylation of dimethylaniline in rabbit liver microsomes is accompanied by inactivation of cytochrome P-450 and the formation of products inhibiting the catalytic activity of non-inactivated cytochrome P-450. Other enzymes and electron carriers of microsomal membrane (cytochrome b5, NADH-ferricyanide reductase, NADPH-cytochrome c and NADPH-cytochrome P-450 reductases) as well as glucose-6-phosphatase were not inactivated in the course of the monooxygenase reactions. Phospholipids and microsomal membrane proteins were also unaffected thereby. Consequently, the changes in the microsomal membrane during cytochrome P-450 dependent monooxygenase system functioning are confined to the inactivation of cytochrome P-450.     

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.     

The NADPH-dependent cytochrome P-450 reduction in liver microsomes of rats of different ages with and without phenobarbital pretreatment.

The activity of NADPH-cytochrome P-450 reductase in liver microsomes of 10- to 60-day-old rats was determined. Neither the half life time of cytochrome P-450 reduction nor the absolute amount of cytochrome P-450 reduced per time unit depend on age. Phenobarbital pretreatment enhances the reduction rate in all age groups. The addition of hexobarbital or ethylmorphine to microsomal suspension accelerates the reduction of cytochrome P-450 in some age groups only. Age differences corresponding to developmental changes in drug-metabolizing activities are not detectable. The NADPH-cytochrome P-450 reductase seems to be not responsible for the age dependence of drug metabolism.     

Purification and characterization of cholesterol 7 alpha-hydroxylase cytochrome P-450 of untreated rabbit liver microsomes

Cytochrome P-450 which catalyzes the 7 alpha-hydroxylation of cholesterol was purified from liver microsomes of untreated rabbits. The minimum molecular weight of the cytochrome P-450 was estimated to be 48,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The preparation contained 7 nmol of cytochrome per mg of protein. The oxidized form of the P-450 showed absorption maxima at 568, 535, and 417 nm, which are characteristic of a low spin hemoprotein, while the reduced form showed maxima at 545 and 413 nm. The carbon monoxide complex of the reduced form showed maxima at 550 and 447 nm. The cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes was reconstituted with the purified P-450, NADPH-cytochrome P-450 reductase, and cytochrome b5. The P-450 catalyzed the 7 alpha-hydroxylation of cholesterol 500 times more efficiently than the starting microsomes. The reconstituted hydroxylase system showed a substantial salt dependency. In the presence of cytochrome b5 the activity was maximum at 0.4 M KCl (4.55 nmol product formed/mg of protein per min), whereas in the absence of cytochrome b5 the activity was marginal (0.65 nmol product formed/mg of protein per min) and inhibited by KCl. Thus, cytochrome b5 stimulated the hydroxylase activity by one order of magnitude. These results indicate that cytochrome b5 is an essential component of the cholesterol 7 alpha-hydroxylase system of untreated rabbit liver microsomes.     

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

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

Immunochemical studies on the contribution of NADPH cytochrome P-450 reductase to the cytochrome P-450-dependent metabolism of arachidonic acid

We have studied the role of NADPH cytochrome P-450 reductase in the metabolism of arachidonic acid and in two other monooxygenase systems: aryl hydrocarbon hydroxylase and 7-ethoxyresorufin-o-deethylase. Human liver NADPH cytochrome P-450 reductase was purified to homogeneity as evidenced by its migration as a single band on SDS gel electrophoresis, having a molecular weight of 71,000 Da. Rabbits were immunized with the purified enzyme and the resulting antibodies were used to evaluate the involvement of the reductase in cytochrome P-450-dependent arachidonic acid metabolism by bovine corneal epithelial and rabbit renal cortical microsomes. A highly sensitive immunoblotting method was used to identify the presence of NADPH cytochrome P-450 reductase in both tissues. We used these antibodies to demonstrate for the first time the presence of cytochrome c reductase in the cornea. Anti-NADPH cytochrome P-450 reductase IgG, but not anti-heme oxygenase IgG, inhibited the NADPH-dependent arachidonic acid metabolism in both renal and corneal microsomes. The inhibition was dependent on the ratio of IgG to microsomal protein where 50% inhibition of arachidonic acid conversion by cortical microsomes was achieved with a ratio of 1:1. A higher concentration of IgG was needed to achieve the same degree of inhibition in the corneal microsomes. The antibody also inhibited rabbit renal cortical 7-ethoxyresorufin-o-deethylase activity, a cytochrome P-450-dependent enzyme. However, the anti-NADPH cytochrome P-450 reductase IgG was much less effective in inhibiting rabbit cortical aryl hydrocarbon hydroxylase. Thus, the degree of inhibition of monooxygenases by anti-NADPH cytochrome P-450 reductase IgG is variable. However, with respect to arachidonic acid, NADPH cytochrome P-450 reductase appears to be an integral component for the electron transfer to cytochrome P-450 in the oxidation of arachidonic acid.     

B-type cytochromes in plasma membranes isolated from rat liver, in comparison with those of endomembranes

Fractions of plasma membranes, Golgi apparatus, endoplasmic reticulum (ER), and nuclear envelope were isolated from rat liver and were characterized by electron microsocpe and biochemical methods. The purity of the fractions was controlled by morphometry and by marker enzyme activities. Amounts of cytochromes b5, P-450, and P-420 were measured, as well as the NADPH- and NADPH-cytochrome c reductase activities. The pigments of the microsomal electron transport system were found in all membrane fractions in relatively high amounts, thus excluding an origin by microsomal contamination. Purified preparations of plasma membrane and Golgi apparatus contained approximately 30% of the cytochrome b5 and cytochrome P-450 + P-420 found in ER membranes. Plasma membranes were also characterized by a high ratio of P-420/450. Degradation of cytochromes P-450 and P-420 was relatively rapid in all fractions, except in the ER. Cytochrome b5 extracted from plasma membranes was spectrophotometrically and enzymatically indistinguishable from ER cytochrome b5. However, immunnlogical characterization with rabbit antibodies against the trypsin-resistant core of microsomal cytochrome b5 showed the presence of at least two types of cytochrome b5 in ER membranes, in contrast to the plasma membranes in which only one of these components was detected. This immunological differentiation also demonstrates that the plasma membrane-bound cytochrome b5 is endogenous to this membrane and does not reflect contamination by ER elements. We conclude that cytochromes b5, P-450, and P-420 are not confined only to ER and nuclear membranes but also occur in signficant amounts in Golgi apparatus and plasma membranes. The findings are discussed in relation to observations of similar redox components in Golgi apparatus, secretory vesicles, and plasma membranes of other cells.     

Hydroxylation of prostaglandin A1 by the microsomes of rabbit intestinal mucosa

Microsomes from rabbit small intestine mucosa were found to catalyze the hydroxylation of PGA1 in the presence of NADPH. The major product was identified as 20-hydroxy PGA1 by using high performance liquid chromatography and gas chromatography-mass spectrometry, and the minor product was assumed to be 19-hydroxy PGA1. The ratio of the former product to the latter was about 24.1. The specific PGA1 omega-hydroxylase activity of small intestine microsomes was comparable to that of liver microsomes, and was significantly higher than those of microsomes from other tissues such as kidney cortex and lung. Microsomes from rabbit colon mucosa also catalyzed the hydroxylation of PGA1 in the presence of NADPH, with the ratio of omega- to (omega-1)-hydroxy PGA1 formed being 33.0. The PGA1 hydroxylase activities of the microsomes from both small intestine and colon were inhibited markedly by carbon monoxide, indicating the participation of cytochrome P-450. A cytochrome P-450 was solubilized from small intestine microsomes, and purified to a specific content of 10.5 nmol of cytochrome P-450/mg of protein. This cytochrome hydroxylated PGA1 at the omega-position with a turnover rate of 38.2 nmol/min/nmol of cytochrome P-450 in the reconstituted system containing cytochrome P-450, NADPH-cytochrome P-450 reductase, cytochrome b5 and phosphatidylcholine. It is suggested that this cytochrome P-450 is specialized for the omega-hydroxylation of PGA1 in small intestine microsomes.     

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 NADPH-cytochrome c reductase from porcine polymorphonuclear leukocytes

NADPH-cytochrome c reductase [NADPH: ferricytochrome oxidoreductase, EC 1.6.2.4] was highly purified from the membrane fraction of porcine polymorphonuclear leukocytes by column chromatographies on DEAE cellulose DE-52, 2',5'-ADP-agarose, Sephacryl S-300, and Bio-gel HTP. Upon sodium dodecyl sulfate polyacrylamide gel electrophoresis, the purified preparation gave a main band with a molecular weight of 80,000. The enzyme contained 0.79 mol of FAD and 0.88 mol of FMN per mol, and was capable of exhibiting a benzphetamine N-demethylation activity in the presence of cytochrome P-450 purified from rabbit liver microsomes and dilauroylphosphatidylcholine, as is the case with liver NADPH-cytochrome P-450 reductase. The cytochrome c reductase activity of the polymorphonuclear leukocytes (PMN) enzyme was precipitated with rabbit anti-guinea pig liver NADPH-cytochrome P-450 reductase IgG followed by addition of guinea pig anti-rabbit IgG antibody. The biochemical and immunological properties of the PMN enzyme so far examined were similar to those of the liver enzyme, although its function in leukocytes has not yet been determined.     

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.     

A form of rabbit liver cytochrome P-450 that catalyzes the 7 alpha-hydroxylation of cholesterol

A form of cytochrome P-450 which comigrates with cytochrome P-450LM4 (molecular weight, 55 000) on SDS-polyacrylamide gel was purified from liver microsomes of cholestyramine-treated rabbits. This form of cytochrome P-450 catalyzed the 7 alpha-hydroxylation of cholesterol with an activity of 37.5 pmol/min per nmol cytochrome P-450 in the reconstituted enzyme system containing cytochrome P-450 and NADPH-cytochrome P-450 reductase. The substrate specificity of this form of cytochrome P-450 was compared with cytochrome P-450LM4 isolated from phenobarbital- and beta-naphthoflavone-treated rabbit liver microsomes. The latter two isoenzymes do not catalyze 7 alpha-hydroxylation of cholesterol, but are more active in O-deethylation of 7-ethoxycoumarin and p-nitrophenetole. Ouchterlony double diffusion revealed cross-reactivity between anti-P-450LM4 (phenobarbital) IgG and cytochrome P-450 isolated from cholestyramine- or beta-naphthoflavone-treated rabbit liver microsomes. A two-dimensional iodinated tryptic peptide fingerprint indicated only minor structural differences among these three cytochrome P-450LM4 preparations.     

Multiple forms of cytochrome P-450 in rabbit colon microsomes

Three cytochrome P-450 preparations, designated as cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction, were separated and purified about 23-, 50-, and 29-fold, respectively, from the cholate extracts of rabbit colon mucosa microsomes. Their specific contents were 1.2, 2.6, and 1.5 nmol of cytochrome P-450 per mg of protein, respectively. Cytochrome P-450ca and cytochrome P-450cb migrated as heme-containing polypeptide bands with molecular weights of about 53,000 and 57,000, respectively, on SDS-polyacrylamide gel electrophoresis. The CO-reduced difference spectra of cytochrome P-450ca, cytochrome P-450cb, and cytochrome P-448c fraction showed maxima at 451, 450, and 449 nm, respectively. Cytochrome P-450ca efficiently catalyzed the omega-hydroxylation of prostaglandin A1 (PGA1) and the omega- and (omega-1)-hydroxylation of caprate, laurate, and myristate in the reconstituted system containing cytochrome P-450ca, NADPH-cytochrome P-450 reductase, cytochrome b5, and phosphatidylcholine. In contrast, cytochrome P-450cb and cytochrome P-448c fraction had no detectable activity toward PGA1 and fatty acids. Both catalyzed aminopyrine and benzphetamine N-demethylation. Cytochrome P-448c fraction also hydroxylated benzo(a)pyrene, and phosphatidylinositol or phosphatidylserine exhibited a stimulatory effect on this activity. The results show that rabbit colon microsomes contain catalytically different cytochrome P-450, one of which is specialized for the omega-oxidation prostaglandins, the others being involved in the metabolism of exogenous compounds such as drugs and polycyclic hydrocarbons.     

Human liver microsomal cytochrome P-450 mephenytoin 4-hydroxylase, a prototype of genetic polymorphism in oxidative drug metabolism. Purification and characterization of two similar forms involved in the reaction

Two forms of cytochrome P-450 (P-450), designated P-450MP-1 and P-450MP-2, were purified to electrophoretic homogeneity from human liver microsomes on the basis of mephenytoin 4-hydroxylase activity. Purified P-450MP-1 and P-450MP-2 contained 12-17 nmol of P-450/mg of protein and had apparent monomeric molecular weights of 48,000 and 50,000, respectively. P-450MP-1 and P-450MP-2 were found to be very similar proteins as judged by chromatographic behavior on n-octylamino-Sepharose 4B, hydroxylapatite, and DEAE- and CM-cellulose columns, spectral properties, amino acid composition, peptide mapping, double immunodiffusion analysis, immunoinhibition, and N-terminal amino acid sequences. In vitro translation of liver RNA yielded polypeptides migrating with P-450MP-1 or P-450MP-2, depending upon which form was in each sample, indicating that the two P-450s are translated from different mRNAs. When reconsituted with NADPH-cytochrome-P-450 reductase and L-alpha-dilauroyl-sn-glyceryo-3-phosphocholine, P-450MP-1 and P-450MP-2 gave apparently higher turnover numbers for mephenytoin 4-hydroxylation than did the P-450 in the microsomes. The addition of purified rat or human cytochrome b5 to the reconstituted system caused a significant increase in the hydroxylation activity; the maximum stimulation was obtained when the molar ratio of cytochrome b5 to P-450 was 3-fold. Rabbit anti-human cytochrome b5 inhibited NADH-cytochrome-c reductase and S-mephenytoin 4-hydroxylase activities in human liver microsomes. In the presence of cytochrome b5, the Km value for S-mephenytoin was 1.25 mM with all five purified cytochrome P-450s preparations, and Vmax values were 0.8-1.25 nmol of 4-hydroxy product formed per min/nmol of P-450. P-450MP is a relatively selective P-450 form that metabolizes substituted hydantoins well. Reactions catalyzed by purified P-450MP-1 and P-450MP-2 preparations and inhibited by anti-P-450MP in human liver microsomes include S-mephenytoin 4-hydroxylation, S-nirvanol 4-hydroxylation, S-mephenytoin N-demethylation, and diphenylhydantoin 4-hydroxylation. Thus, at least two very similar forms of human P-450 are involved in S-mephenytoin 4-hydroxylation, an activity which shows genetic polymorphism.     

Biosynthesis of cytochrome P-450 on membrane-bound ribosomes and its subsequent incorporation into rough and smooth microsomes in rat hepatocytes

Intracellular sites of synthesis of cytochrome P-450 and the subsequent incorporation of it into membrane structures of the endoplasmic reticulum (ER) in rat hepatocytes have been studied using an antibody monospecific for phenobarbital-inducible cytochrome P-450. The cytochrome is synthesized mainly on the "tightly bound" type of membrane-bound ribosomes whose release from the membrane requires treatment with puromycin in a high salt buffer (500 mM KCI, 5mM MgCl2, and 50 mM Tris-HCL [pH 7.5]). Subsequently the cytochrome is incorporated directly into the rough ER membranes with its major part exposed to the outer surface to the membrane and accessible to proteolytic enzymes added externally. The newly synthesized molecules, which appeared first in the rough membrane, are translocated to the smooth membrane, and are then distributed evenly between the two types of microsomeal membranes in approximately 1 h. Administration of cycloheximide, an inhibitor of protein biosynthesis, did not significantly inhibit the transfer of the enzyme from the rough to the smooth ER. It is suggested, therefore, that the translocation of the newly synthesized cythochrome P-450 between the rough and smooth microsomes is mainly due to the lateral movement of the molecules in the plane of the membranes rather than to the attachment and detachment of the ribosomes on the microsomal membranes after the ribosomal cycle for protein synthesis.     

Hydroxylation of prostaglandins by inducible isozymes of rabbit liver microsomal cytochrome P-450. Participation of cytochrome b5

The hydroxylation of prostaglandin (PG) E1, PGE2, and PGA1 was investigated in a reconstituted rabbit liver microsomal enzyme system containing phenobarbital-inducible isozyme 2 or 5,6-benzoflavone-inducible isoenzyme 4 of P-450, NADPH-cytochrome P-450 reductase, phosphatidylcholine, and NADPH. Significant metabolism of prostaglandins by isozyme 2 occurred only in the presence of cytochrome b5. Under these conditions, PGE1 hydroxylation was linear with time (up to 45 min) and protein concentration, and maximal rates were obtained with a 1:1:2 molar ratio of reductase: cytochrome b5:P-450LM2. Moreover, P-450LM2 catalyzed the conversion of PGE1, PGE2, and PGA1 to the respective 19- and 20-hydroxy metabolites in a ratio of about 5:1, and displayed comparable activities toward the three prostaglandins based on the total products formed in 60 min. Apocytochrome b5 or ferriheme could not substitute for intact cytochrome b5, while reconstitution of apocytochrome b5 with ferriheme led to activities similar to those obtained with the native cytochrome. Isozyme 4 of P-450 differed markedly from isozyme 2 in that it catalyzed prostaglandin hydroxylation at substantial rates in the absence of cytochrome b5, was regiospecific for position 19 of all three prostaglandins, and had an order of activity of PGA1 greater than PGE1 greater than PGE2. P-450LM4 preparations from untreated and induced animals had similar activities with PGE1 and PGE2, respectively. Addition of cytochrome b5 resulted in a 20 to 30% increase in the rate of PGE1 hydroxylation and an appreciably greater enhancement in the extent of all the P-450LM4-catalyzed reactions, the stimulation being greatest with PGE2 (3-fold) and least with PGA1 (1.6-fold). Cytochrome b5 was thus required for maximal metabolism of all three prostaglandins, but did not alter the regiospecificity or the order of activity of P-450 isozyme 4 with the individual substrates. In the presence of cytochrome b5, the prostaglandin hydroxylase activities of isozyme 4 were two to six times higher than those of isozyme 2.     

Analysis of debromination of 1,2-dibromoethane by cytochrome P-450-linked hydroxylation systems as observed by bromide electrode

Debromination of 1,2-dibromoethane (DBE) by a rabbit liver microsomal preparation and a reconstituted cytochrome P-450 enzyme system was investigated. The reaction was performed in our newly constructed reaction vessel, in which a bromide electrode was installed. During the reaction, the liberated bromide ion was continuously measured by the bromide electrode, and the amount was recorded. In the microsomal preparation, the DBE-debromination rate per nmol cytochrome P-450 was enhanced by phenobarbital-pretreatment of rabbits compared with the untreated microsomes, whereas it was diminished by 3-methylcholanthrene-pretreatment. The debromination reaction was reconstituted in a purified enzyme system containing phenobarbital-inducible rabbit liver microsomal cytochrome P-450 (P-450PB), NADPH-cytochrome P-450 reductase, and NADPH. The optimum conditions required the presence of dilauroylphosphatidylcholine and cytochrome b5. Cytochrome b5 was found not to be an obligatory component for the DBE-debromination in the reconstituted system, but it stimulated the activity about 3.4-fold. Preincubation of the reconstituted mixture with guinea pig anti-cytochrome P-450PB antiserum markedly inhibited the debromination reaction.     

Alcohol-inducible cytochrome P-450IIE1 lacking the hydrophobic NH2-terminal segment retains catalytic activity and is membrane-bound when expressed in Escherichia coli

We have expressed in Escherichia coli a cDNA encoding rabbit liver cytochrome P-450IIE1, the ethanol-inducible P-450. The expressed P-450 is located primarily in the bacterial inner cell membrane and comprises 3% of the E. coli total membrane protein. The partially purified cytochrome exhibits a reduced CO difference spectrum with a maximum at 452 nm, characteristic of P-450IIE1, and solubilized membranes or partially purified P-450 preparations reconstituted with NADPH-cytochrome P-450 reductase and phosphatidylcholine catalyze the deethylation of N-nitrosodiethylamine with a turnover number equal to that of purified liver P-450IIE1 (approximately 4.5 nmol/min/nmol of P-450). A modified IIE1 cDNA that encodes a protein lacking amino acids 3-29, a proposed membrane anchor for cytochrome P-450, was also expressed in E. coli and, unexpectedly, the shortened protein was also found to be predominantly located in the bacterial inner membrane rather than the cytosol. Like the full-length protein, this truncated cytochrome has a reduced CO difference spectrum characteristic of P-450IIE1 and is fully active in the deethylation of N-nitrosodiethylamine. These results demonstrate that the NH2-terminal hydrophobic segment is not solely responsible for attachment to the membrane and evidently is not required for proper protein folding or catalytic activity.     

Isolation by ion-exchange high performance liquid chromatography of rabbit liver cytochrome P-450 with regioselectivity for omega-hydroxylation of prostaglandins

Previous studies suggested that rabbit liver microsomes contain cytochrome P-450 monooxygenase(s) with low affinity for (omega-1)-hydroxylation and high affinity for omega-hydroxylation of prostaglandins (Theoharides, A. D., and Kupfer, D. (1981) J. Biol. Chem. 256, 2168-2175). The current investigation describes the isolation from livers of untreated rabbits of a cytochrome P-450 catalyzing, with regioselectivity, the omega-hydroxylation of prostaglandins E1 and E2. The isolation of the enzyme involved enrichment of the omega-hydroxylase activity by polyethylene glycol 8000 fractionation, followed by ion-exchange high performance liquid chromatography. Based on Mr of 59,000-60,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the isolated enzyme is referred to as P-450 form 7. This P-450 exhibits a low spin spectrum (lambda max = 417 nm) and a difference spectrum of the CO-reduced complex versus reduced (lambda max = 451 nm). For catalytic activity, the P-450 form 7 was reconstituted with NADPH-P-450 reductase, cytochrome b5, and lipid. There was no activity in the absence of the reductase, and deletion of cytochrome b5 yielded a minimal amount of product (heme could not substitute for cytochrome b5), demonstrating an absolute requirement for these components.