Incorporation of heme to microsomal cytochrome P-450 in the absence of protein biosynthesis

Determination of the heme and protein portions of phenobarbital (PB)-inducible and 3-methylcholanthrene inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), in the liver microsomes of drug-treated animals indicated the presence of 20-30% of apo-cytochrome P-450 in both cases. Inhibition of protein synthesis by cycloheximide injection to the rats did not significantly inhibit the incorporation of delta-amino[14C]levulinic acid (ALA) into the heme of P-450(PB-1) or P-450(MC-1) in the liver, indicating that the heme incorporation into microsomal cytochrome P-450 is not tightly coupled with the synthesis of the apo-cytochrome. When heme-labeled cytosol prepared from [14C]ALA-injected rats was incubated with non-radioactive microsomes in vitro, a significant amount of labeled heme was incorporated into microsomal P-450(PB-1), whereas the incorporation into P-450(MC-1) was much less. The in vitro transfer of heme from cytosol to microsome-bound cytochrome P-450 was stimulated by the addition of an NADPH-generating system to the incubation mixtures, and inhibited when the microsomes were solubilized with sodium cholate and Emulgen-913. Although the in vitro incubation of heme-labeled microsomes with non-radioactive cytosol resulted in some release of labeled heme from the microsomes, no reversible transfer of heme between cytochrome P-450 molecules bound to separate microsomal vesicles was detected when heme-labeled microsomes were incubated with non-radioactive microsomes in the presence and absence of cytosol.     

Isolation of a subfraction of rough endoplasmic reticulum closely associated with mitochondria : Evidence for its role in cytochrome P450 synthesis

A subfraction of rough endoplasmic reticulum (RER) structurally associated with mitochondria (mito-RER complexes) was isolated from crude nuclear fractions of rat liver homogenate. When apocytochrome P450 synthesis (which presumably occurs in RER) and mitochondrial heme synthesis was dissociated by concomitant treatment of rats with phenobarbital and cobaltous chloride, apocytochrome P450 accumulated predominantly in mito-RER complexes. These data suggest that cytochrome P450 synthesis requires structural interaction of mitochondria and RER.     

3-(Trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine photolabels a substrate-binding site of rat hepatic cytochrome P-450 form PB-4

Hepatic microsomes isolated from untreated male rats or from rats pretreated with phenobarbital (PB) or 3-methylcholanthrene (3-MC) were labeled with the hydrophobic, photoactivated reagent 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine ([125I]TID). [125I]TID incorporation into 3-MC- and PB-induced liver microsomal protein was enhanced 5- and 8-fold, respectively, relative to the incorporation of [125I]TID into uninduced liver microsomes. The major hepatic microsomal cytochrome P-450 forms inducible by PB and 3-MC, respectively designated P-450s PB-4 and BNF-B, were shown to be the principal polypeptides labeled by [125I]TID in the correspondingly induced microsomes. Trypsin cleavage of [125I]TID-labeled microsomal P-450 PB-4 yielded several radiolabeled fragments, with a single labeled peptide of Mr approximately 4000 resistant to extensive proteolytic digestion. The following experiments suggested that TID binds to the substrate-binding site of P-450 PB-4. [125I]TID incorporation into microsomal P-450 PB-4 was inhibited in a dose-dependent manner by the P-450 PB-4 substrate benzphetamine. In the absence of photoactivation, TID inhibited competitively about 80% of the cytochrome P-450-dependent 7-ethoxycoumarin O-deethylation catalyzed by PB-induced microsomes with a Ki of 10 microM; TID was a markedly less effective inhibitor of the corresponding activity catalyzed by microsomes isolated from uninduced or beta-naphthoflavone-induced livers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Suitability of L-[35S]methionine for studying the biosynthesis of the polypeptides of mouse liver endoplasmic reticulum membrane fractions in vivo.

The use of L-[35S]methionine (500-700 Ci/mmol (1 Ci = 37 GBq) for labelling the polypeptides of liver rough (R) and smooth (S)endoplasmic reticulum (ER) membrane fractions in vivo was studied. Adult mice were injected intraperitoneally with 400 muCi of the isotope and killed at various times (2'min to 24 h) thereafter. RER and SER fractions were prepared, stripped of ribosomes, and treated with Triton X-100 to remove intravesicular contents. Sufficient radioactivity was present in individual aliquots (75 microgram protein) of the ER membrane fractions to permit their analysis by fluorography after separation by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. By 3 min, although the majority of the labelled components were of intravesicular origin, some 12 membrane polypeptides were labelled in the RER fraction (including one corresponding in migration to cytochrome P-450); some 6 of these latter polypeptides were labelled to a lesser degree in the SER membrane fraction at this time. By 5 min, the patterns of radioactive polypeptides of the RER and SER fractions (including both membrane and intravesicular components) were identical. By 7 min, some 28 labelled membrane polypeptides were detectable in the total microsomal membrane. Analysis of the 24-h samples revealed that all the membrane polypeptides seen by staining with Coomassie blue were visualised by fluorography. Other studies revealed the applicability of the approach used for producing highly labelled cell sap and serum proteins. The overall results demonstrate the suitability of L-[35S]methionine administered in vivo for producing mouse liver ER membrane polypeptides of relatively high radioactivity and are consistent with a rapid conversion of RER to SER by ribosome detachment or membrane flow.     

Inhibition of overall protein and RNA synthesis as a mechanism for the tunicamycin induced decrease in cytochrome P-450 in rat hepatocytes

In rat hepatocytes maintained in culture, cytochrome P-450 and NADPH cytochrome c reductase activities were decreased by tunicamycin in a dose and time dependent fashion. The effect of tunicamycin was mainly due to inhibition of protein synthesis. Tunicamycin decreased L-[35S] methionine incorporation into many proteins, including a 52 kDa cytochrome P-450 isozyme. Tunicamycin also reduced RNA synthesis. These results indicate that tunicamycin decreased cytochrome P-450 levels in hepatocytes by inhibiting protein and RNA synthesis.     

Metabolism of benzo[c]phenanthrene by rat liver microsomes and by a purified monooxygenase system reconstituted with different isozymes of cytochrome P-450

Metabolism of the environmental pollutant and weak carcinogen benzo[c]-phenanthrene (B[c]Ph) by rat liver microsomes and by a purified and reconstituted cytochrome P-450 system is examined. B[c]Ph proved to be one of the best polycyclic aromatic hydrocarbon substrates for rat liver microsomes. It is metabolized by microsomes from control rats and by rats treated with phenobarbital or 3-methylcholanthrene at 3.9, 4.2 and 7.8 nmol/nmol cytochrome P-450/min, respectively. Principal metabolites are dihydrodiols along with small amounts (less than 10%) of phenols. The K-region 5,6-dihydrodiol is the major metabolite and accounts for 77-89% of the total metabolites. The 3,4-dihydrodiol with a bay-region 1,2-double bond is formed in much smaller amounts and accounts for only 6-17% of the total metabolites, the highest percentage being formed by microsomes from control rats. Highly purified monooxygenase systems reconstituted with cytochrome P-450a, P-450b and P-450c and epoxide hydrolase form predominantly the 5,6-dihydrodiol (95-97% of total metabolites) and only a small percentage of the 3,4-dihydrodiol (3-5% of total metabolites). The 3,4-dihydrodiol is formed with higher enantiomeric purity by microsomes from 3-methylcholanthrene-treated rats (88%) than by microsomes from control rats (78%) or phenobarbital-treated rats (60%). In each case the (3R,4R)-enantiomer predominates. B[c]Ph 5,6-dihydrodiol formed by all three microsomal preparations is nearly racemic.     

Regulation of cytochrome P-450p by phenobarbital and phenobarbital-like inducers in adult rat hepatocytes in primary monolayer culture and in vivo

Treatment of rats with phenobarbital increases the hepatic concentration of P-450p, a form of cytochrome P-450 believed to be controlled primarily by a mechanism that stereospecifically recognizes glucocorticoids like dexamethasone and anti-glucocorticoids like pregnenolone-16 alpha-carbonitrile [Schuetz, E.G., & Guzelian, P.S. (1984) J. Biol. Chem. 259, 2007]. To test the possibility that phenobarbital induces P-450p indirectly by increasing the availability of endogenous glucocorticoids in the liver, we added phenobarbital and phenobarbital-like inducers to primary monolayer cultures of adult rat hepatocytes incubated in serum-free medium without glucocorticoids and found stimulated de novo synthesis of P-450p measured as increased incorporation of [3H]leucine into immunoprecipitable P-450p protein. With some of the inducers, notably the organochlorine pesticides chlordane and trans-nonachlor, there was a greater accumulation of P-450p measured on quantitative immunoblots than could be accounted for by the increase in P-450p synthesis. "Pulse-chase" experiments confirmed that these compounds significantly lengthen the half-life of P-450p up to 60 h as compared to the values in control (11 h) or dexamethasone-treated (10 h) cultures. Treatment of rats with chlordane, trans-nonachlor, or other cyclodiene organochlorine pesticides confirmed that these agents increase the concentration of P-450p in liver microsomes analyzed on immunoblots of two-dimensional electrophoretic gels. The time courses of induction in trans-nonachlor-treated rats of P-450p protein and of P-450PB proteins induced by phenobarbital were similar as were the amounts of P-450PB mRNA and P-450p mRNA measured by hybridization to cloned cDNA probes. However, analysis of structure-activity relationships among polychlorinated biphenyls revealed that isomers with two ortho chlorinated positions maximally induced P-450PB whereas isomers with three and four ortho chlorines maximally induced P-450p in rats and in hepatocyte culture, respectively. We conclude that P-450p is induced by the phenobarbital class of inducers through direct contact with the hepatocytes involving decreased degradation of the protein and stimulation of its synthesis in a manner similar but not identical with that of P-450PB.     

Isolation and characterization of rough endoplasmic reticulum associated with mitochondria from normal rat liver

A subfraction of rough endoplasmic reticulum (RER) characterized by its close association with mitochondria (MITO) was isolated from low speed pellets of normal rat liver homogenate under defined ionic conditions. This fraction enriched in MITO-RER complexes contained 20% of cellular RNA, 20% of glucose-6-phosphatase and 47% of cytochrome c oxidase activities. Morphologically, the isolated MITO-RER complexes closely resembled physiological associations between the two organelles commonly seen in intact liver. Partial dissociation of RER from mitochondria of the MITO-RER fraction was achieved by either EDTA (0.5 mM) or by hypotonic/hypertonic treatment of MITO-RER complexes. With the latter procedure approx. 70% of RER (RERmito) with 50% of ribosomes still attached could be separated from the inner compartments of mitochondria. This RERmoto exhibited a higher glucose-6-phosphatase activity than RER isolated as rough microsomes from the postmitochondrial supernatant. Isopycnic centrifugation on linear metrizamide gradients revealed that the mitochondria-associated part of RER corresponds to the high density, ribosome-rich subfraction of rough microsomes isolated in cation-free sucrose solution. The combined data demonstrate that a morphologically and biochemically distinct portion of RER is associated with mitochondria and support the concept of considerable intracellular heterogeneities in distribution of enzymes and enzyme systems along the lateral plane of the endoplasmic reticulum membrane system.     

Effects of acetylenic and olefinic pyrenes upon cytochrome P-450 dependent benzo[a]pyrene hydroxylase activity in liver microsomes

1-Ethynylpyrene, trans-, & cis-1-(2-bromovinyl)pyrene, methyl 1-pyrenyl acetylene, and phenyl 1-pyrenyl acetylene are substrates for cytochrome P-450 dependent monooxygenases and also inhibitors of cytochrome P-450 dependent benzo[a]pyrene hydroxylase activities in liver microsomes from 5,6-benzoflavone or phenobarbital pretreated rats. 1-Ethynylpyrene, trans-1-(2-bromovinyl)pyrene, and methyl 1-pyrenyl acetylene cause a mechanism based inhibition (suicide inhibition) of the benzo[a]pyrene hydroxylase activities in microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, while cis-1-(2-bromovinyl)pyrene only causes suicide inhibition of the hydroxylse activities in the 5,6-benzoflavone induced microsomes and phenyl 1-pyrenyl acetylene does not cause a detectable suicide inhibition of these activities in either type of microsome. Incubation with NADPH and 1-ethynylpyrene, trans-, or cis-1-(2-bromovinyl)pyrene causes a loss of the P-450 content in the microsomes from 5,6-benzoflavone or phenobarbital pretreated rats, but incubations with methyl 1-pyrenyl acetylene or phenyl 1-pyrenyl acetylene did not cause a loss of the P-450 content of either microsomal preparation.     

Phenobarbital-type induction of cytochrome P-450 in liver microsomes by perfluorodecalin

Cytochrome P-450 induction in hepatic microsomes after injections of rats with a fluorocarbon emulsion containing perfluorodecalin was studied in comparison with phenobarbital and methylcholanthrene type inductions. It was shown that perfluorodecalin injection as well as the phenobarbital one cause an increase in the cytochrome P-450 content, NADPH-cytochrome c reductase activity, the rates of benzphetamine N-demethylation and aldrin epoxidation in the microsomes. Using the Ouchterlony double immunodiffusion test with antibodies against cytochrome P-450b, an immunological identity of cytochrome P-450 isoforms during perfluorodecalin and phenobarbital inductions was shown. Upon "rocket" immunoelectrophoresis the recovery of cytochrome P-450 which is immunologically indistinguishable from cytochrome P-450b was approximately 72% in perfluorodecalin-induced microsomes. The activity of benzphetamine demethylase and aldrin epoxidase was inhibited by antibodies against cytochrome P-450b. These results suggest that in rat hepatic microsomes perfluorodecalin induces the cytochrome P-450 isoform whose immunological properties and substrate specificity correspond to those of phenobarbital-type cytochrome P-450.     

Induction of NADPH-Cytochrome P-450 (c) Reductase in Wounded Tissues from Helianthus tuberosus Tubers

Cytochrome P-450 is not self-sufficient for the catalysis ofmonooxygenase reaction but requires NADPH and NADPH-cytochromeP-450 (c) reductase. The activity of NADPH-cyto-chrome P-450reductase was strongly enhanced by wounding and aging in Jerusalemartichoke (Helianthus tuberosus L.) tuber tissues. This stimulationwas correlated with the synthesis of the enzyme protein basedon i) quantitation of the reductase protein by Western blotting,ii) incorporation of [³⁵S]methionine into the immunoprecipitableenzyme and iii) an increase in translatable mRNA for the reductasein a cell free system. (Received April 9, 1990; Accepted September 12, 1990)     

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.     

Studies on the interaction of furan with hepatic cytochrome P-450

In vitro incubation of rat liver micro-somes with [¹⁴C]-furan in the presence of NADPH resulted in the covalent incorporation of furan-derived radioactivity in microsomal protein. Compared to microsomes from untreated rats a two- to threefold increase in binding was observed with microsomes from phenobarbital-treated rats and a four- to five-fold increase was observed with microsomes from rats pretreated with imidazole or pyrazole. Covalent binding was reduced with microsomes from rats pretreated with β-naphthoflavone. Chemicals containing an amine group (semicarbazide), those in which the amine group is blocked but have a free thiol group (N-acetylcysteine), and those which have both an amine and a thiol group (glutathione) effectively blocked binding of [¹⁴C]-furan to microsomal protein. A decrease in cytochrome P-450 (P-450) content and decreases in the activities of P-450-dependent aniline hydroxylase, 7-ethoxycoumarin-O-deethylase (BCD), and 7-ethoxyresorufin-O-deethylase (ERD) was observed 24 hours after a single oral administration of 8 or 25 mg/kg of furan, suggesting that the reactive intermediate formed during P-450 catalyzed metabolism could be binding with nucleophilic groups within the P-450. In vitro studies indicated a significant decrease in the activity of aniline hydroxylase in pyrazole microsomes and BCD in phenobarbital microsomes without any significant change in the CO-binding spectrum of P-450 or in the total microsomal heme content, suggesting that furan inhibits the P-450s induced by PB and pyrazole. An almost equal distribution of furan-derived radioactivity in the heme and protein fractions of the CO-binding particles after In vitro treatment of microsomes with furan suggests binding of furan metabolites with heme and apoprotein of P-450, and, probably, due to this interaction, furan is acting as a suicide inhibitor of P-450.     

Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.     

Differential expression and function of three closely related phenobarbital-inducible cytochrome P-450 isozymes in untreated rat liver

The levels of expression of cytochromes P-450b and P-450e (both inducible by phenobarbital (PB) and differing by only 14 of 491 amino acids) in liver microsomes from untreated male rats were separately quantitated by Western blotting with a polyclonal antibody raised against P-450b that is equally effective against P-450e (anti P-450b/e). A protein with mobility identical to P-450e was detected in all microsomal samples. Microsomes from uninduced livers of individual male rats from five different strains exhibited only minor interstrain and interindividual variability in the expression of P-450e (17 +/- 5 pmol P-450e/mg microsomal protein) with the exception of the Brown Norway strain (8.5 +/- 0.5 pmol P-450e/mg). Expression of P-450b varied widely from undetectable levels (less than 2 pmol/mg) in most Sprague-Dawley rats to about 50% of P-450e levels in Fischer and Brown Norway strains. Anti P-450b/e inhibited total metabolism of 7,12-dimethylbenz[a]anthracene (DMBA) by uninduced microsomes, to an extent dependent on rat strain (15-30%), predominantly through inhibition of formation of 12-hydroxymethyl-7-methyl BA (12HOMMBA) (65-85%), the major metabolite of purified P-450e. A specific activity for P-450e-dependent DMBA metabolism was calculated from four sets of microsomes where the P-450b content was either undetectable or very low (0.7-1.0 nmol/nmol P-450e/min-1). Comparable calculated activities were, however, obtained from other untreated rat liver microsomes where P-450b levels were significant. Polymorphism in P-450b was detected but did not affect total P-450b expression or the sensitivity of DMBA metabolism to anti P-450b/e. A fourth band of greater mobility than P-450b (apparent Mr less than 50,000), was also recognized by anti P-450b/e. The intensity of this band did not vary among individual rats or among the different strains and therefore did not correlate with the sensitivity of microsomal DMBA metabolism to anti P-450b/e. A monoclonal antibody (MAb) against P-450b (2-66-3) recognized P-450's b, b2, and e on Western blots but did not react with this higher mobility band. MAb 2-66-3 and two other MAbs produced against P-450b inhibited 12-methylhydroxylation of DMBA by untreated rat liver microsomes to the same extent as anti P-450b/e. Following PB induction, P-450b was induced to about double the level of P-450e in most rat strains examined.(ABSTRACT TRUNCATED AT 400 WORDS)     

Accelerated hepatic haem catabolism in the selenium-deficient rat.

1. Hepatic microsomal cytochrome P-450 concentrations are lower in selenium-deficient rats treated with phenobarbital for 4 days than in similarly treated control rats. 2. No defect in haem synthesis was found on the basis of measurements of delta-aminolaevulinate synthase (EC 2.3.1.37), delta-aminolaevulinate dehydratase (EC 4.2.1.24) and ferrochelatase (EC 4.99.1.1) activities, and urinary excretion of delta-aminolaevulinate, porphobilinogen, uroporphyrin and coproporphyrin. 3. No defect in apo-(cytochrome P-450) separated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 4. An increase in haem catabolism was found. An 8-fold increase in hepatic microsomal haem oxygenase (EC 1.14.99.3) activity occurred in selenium-deficient rats after phenobarbital treatment, compared with a less than 2-fold increase in control rats. Also excretion of 14CO in the breath after administration of delta-amino[5-14C]laevulinate was greater by phenobarbital-treated selenium-deficient rats than by similarly treated controls. 5. These studies demonstrate that the defective induction of cytochrome P-450 by phenobarbital in selenium-deficient rats is accompanied by increased haem catabolism. This could be due to increased breakdown of cytochrome P-450 or to catabolism of haem before it attaches to the apo-cytochrome. The role of selenium in stabilizing cytochrome P-450 and/or in protecting haem from breakdown remains to be determined.     

Lipid composition and turnover of rough and smooth microsomal membranes in rat liver

Subfractions of rat liver microsomes (rough, smooth I, and smooth II), isolated in a cation-containing sucrose gradient system, were analyzed. After removal of adsorbed and luminal protein, these subfractions had the same phospholipid/protein ratio, about 0.40. Both the classes and the relative amounts of phospholipids were similar in the three subfractions, but the relative amounts of neutral lipids (predominantly free cholesterol and triglycerides) were higher in smooth I and especially in smooth II than in rough microsomes. Various pieces of evidence indicate that the neutral lipids are tightly bound to the membranes. Glycerol-(3)H was incorporated into the phospholipids of the rough and smooth I microsomes significantly faster than into those of the smooth II membranes; (32)P incorporation followed a similar but less pronounced pattern. Acetate-(3)H was incorporated into the free cholesterol of smooth I microsomes only half as fast as into the other two subfractions. Injection of phenobarbital increased the cellular phospholipid and neutral lipid content in the rough and smooth I, but not in the smooth II microsomes. Consequently, the neutral lipid/phospholipid ratio of all three subfractions remained unchanged after phenobarbital treatment. It is concluded that the membranes of the rough and the two smooth microsomal subfractions from rat liver have a similar phospholipid composition, but are dissimilar in their neutral lipid content and in the incorporation rate of precursors into membrane lipids.     

Rabbit nasal cytochrome P-450 NMa has high activity as a nicotine oxidase

Rabbit nasal olfactory and respiratory microsomes demonstrate high activity toward [3H]-(S)-nicotine, with specific activities of 22.2 and 6.5 nmol/min/mg protein, respectively. The major metabolite produced is (S)-nicotine delta 1'; 5'-iminium ion, with lesser amounts of nornicotine and the N'-oxide. Reconstitution of the rabbit nasal microsomal system with cytochromes P-450 NMa and NMb indicated that only P-450 NMa has significant activity toward nicotine, and the metabolite profile and turnover are similar to that observed with nasal microsomes. The low Km (35 microMs) and high Vmax (28 min-1) suggest that a significant portion of inhaled nicotine is metabolized by nasal tissues in the rabbit.     

Constitutive testosterone 6 beta-hydroxylase in rat liver

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

Limitations on the metyrapone assay for the major phenobarbital inducible form of cytochrome P-450

Limitations on the determination of the concentration of the major phenobarbital inducible form of cytochrome P-450 (P-450b) in hepatic microsomes by the metyrapone assay of Luu-The $\text{et al.}$ (1) are reported. Compounds which bind to the Type I, II and IR binding sites, or convert cytochrome P-450 to P-420, decrease the apparent concentration of cytochrome P-450b by 20 to 100% in hepatic microsomes from untreated and pregnenolone-16α-carbonitrile or phenobarbital treated rats. It is calculated that errors of greater ca. 40% in the concentration of cytochrome P-450b can arise in the presence of appreciable quantities of the major pregnenolone-16α-carbonitrile or polycyclic hydrocarbon inducible forms of cytochrome P-450.