Strain differences in the maintenance of cytochrome P-450 and mixed-function-oxidase activities in cultured rat hepatocytes. Effect of prostaglandins.

The mixed-function-oxidase (MFO) activities, ethoxyresorufin and pentoxyphenoxazone O-dealkylase, of cultured Hooded-Lister(HL)-rat hepatocytes declined rapidly during 72 h of culture, whereas in Sprague-Dawley(SD)-rat hepatocytes the MFO activities increased between 24 and 72 h in culture. Cytochrome P-450 content declined at the same rate in both HL- and SD-rat hepatocyte cultures. NADPH:cytochrome c reductase and NADH:cytochrome b5 reductase were more stable in SD- than in HL-rat hepatocyte cultures. 16,16-Dimethylprostaglandins E2 and F2 alpha improved the maintenance of cytochrome P-450 content, MFO activity and NADPH:cytochrome c reductase in the HL-rat hepatocyte cultures. In SD-rat hepatocytes, the prostaglandins had no effect on cytochrome P-450 content or NADPH:cytochrome c reductase activity, whereas they prevented the increase observed in MFO activities between 24 and 72 h after culture.     

Induction and regulation of cytochrome P450 K-5 (lauric acid hydroxylase) in rat renal microsomes by starvation

The effects of starvation on rat renal cytochrome P-450s were studied. The content of spectrally measured cytochrome P-450 in the renal microsomes of male rats increased 2-fold with 72 h starvation, but cytochrome b5 and NADPH-cytochrome P-450 reductase were not induced. 7-Ethoxycoumarin O-dealkylation and aniline hydroxylation activities of the renal microsomes of control male rats were very low but were induced 2.5-3-fold by 72 h starvation. Aminopyrine N-demethylation and lauric acid hydroxylation activities were induced 1.5-2-fold by 72 h starvation. The changes in catalytic activities suggested that the contents of individual cytochrome P-450s in the renal microsomes were altered by starvation. The contents of some cytochrome P-450s were measured by Western blotting. P450 DM (P450IIE1), a typical form of cytochrome P-450 induced by starvation in rat liver, was barely detected in rat kidney and was induced 2-fold by 72 h starvation. P450 K-5, a typical renal cytochrome P-450 and lauric acid hydroxylase, accounted for 81% of the spectrally measured cytochrome P-450 in the renal microsomes of control male rats and was induced 2-fold by 72 h starvation. P450 K-5 was not induced in rat kidney by treatment with chemicals such as acetone or clofibrate. The renal microsomes of male rats contained 6-times as much P450 K-5 as those of female rats. These results suggest that P450 K-5 is regulated by an endocrine factor.     

Differential stability of drug-metabolizing enzyme activities in primary rat hepatocytes, cultured in the absence or presence of dexamethasone

The effects of primary hepatocyte culture on the rat cytochrome P450-dependent monooxygenase system and several conjugating enzyme activities were examined using a culture system similar to those used for evaluation of chemicals as potential genotoxins. Cytochrome P450 and cytochrome b5 contents progressively decreased throughout the 72-h culture period to less than 25% of initial values, whereas cytochrome P450 reductase rapidly decreased by 50% during attachment, but then remained stable. Cytochrome P450-dependent testosterone hydroxylase activities decreased more rapidly in culture than did cytochrome P450 content reaching less than 50% of attachment levels by 24 h. Cytochrome P450IIIA immunoreactive protein decreased at a similar rate to testosterone-6 beta-hydroxylase. Activated UDP-glucuronyltransferase activities towards 1-naphthol and testosterone declined more slowly over the 72 h than cytochrome P450 and remained at 50-60% of initial values at 72 h. UDP-glucuronyltransferase activity towards digitoxigenin monodigitoxoside (DIG) did not decrease during culture. Glutathione-S-transferase and sulfotransferase activities also declined during the 72 h at rates which appeared to be isozyme-dependent. Addition of 1 microM dexamethasone (DEX) to the culture medium increased UDP-glucuronyltransferase activity towards DIG, cytochrome P450 reductase and testosterone-6 beta-hydroxylase activities up to 2.5-, 2.0- and 7-fold, respectively and induced cytochrome P450IIIA immunoreactive protein(s) in the hepatocytes after 24 and 48 h of culture; DEX was less effective at the 72 h time-point. DEX treatment also significantly accelerated the decreases in glutathione-S-transferase activities and in sulfotransferase activities towards 1-naphthol and estrone. Thus, it appears that primary rat hepatocytes cultured under standard conditions, not only rapidly lose their monooxygenase capabilities, but also some of their capacity for conjugation. Furthermore, the use of DEX in cell culture medium to enhance cell survival does not maintain total drug-metabolizing enzyme capability, but appears to transiently and selectively increase expression of certain isozymes at the expense of others.     

Effects of energy and/or protein restriction on hepatic microsomal mixed function oxidase in male broiler chicks

1. Effect of energy and/or protein intake on a mixed function oxidase system (MFO) in hepatic microsomes was studied in male broiler chicks. 2. Contents of cytochrome P-450 and b5 in 72 hr starvation were larger than those in 12 and 36 hr starvations. 3. Reduction of energy and protein intake did not change the MFO, except cytochrome P-450. 4. Reduction of energy intake under the same protein intake increased the cytochrome P-450 content and aminopyrine N-demethylase activity. An increase in protein intake under the same energy intake also increased the cytochrome P-450 content.     

Influence of oxygen on the microsomal electron transport system in Saccharomyces cerevisiae

The influence of oxygen on the level of microsomal electron transport chain components has been studied during the growth of Saccharomyces cerevisiae. Enzyme activities and cytochrome content were assayed in microsomal fractions prepared from a protoplast lysate free from mitochondrial contamination. It was found that the cytochrome P-450 and cytochrome b5 content, to get her with the NADPH-cytochrome (P-450)-reductase and NADH-cytochrome (b5)-reductase activities, were increased in the cells as the pO2 of the medium was decreasing. At the same time an increase in the membrane surface of the endoplasmic reticulum can be observed.     

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.     

Zonal distribution of cytochromes P-450 and related enzymes of bovine adrenal cortex--quantitative assay of concentrations and total contents

The zona glomerulosa, zona fasciculata, zona reticularis, and medulla were separated from bovine adrenal glands and cytochromes P-450 and related enzymes in each zone were investigated immunochemically by Western blotting using antisera from chickens or rabbits against cytochromes P-450scc, P-450(11)beta, P-450s21, and b5, NADH-cytochrome b5 reductase, NADPH-cytochrome P-450 reductase, NADPH-adrenodoxin reductase, and adrenodoxin. Concentrations of cytochrome P-450(11)beta, NADPH-cytochrome P-450 reductase, and cytochrome b5 per milligram of protein of homogenate were higher in the zona glomerulosa than in the other zones; the levels of the other components were higher in the zona fasciculata. The total enzyme content of all components was the highest in the zona fasciculata. The amount of adrenodoxin was about 10 times that of NADPH-adrenodoxin reductase in each zone.     

Effects of Hormones on Changes in Cytochrome P-450, Prolyl Hydroxylase,and Glycerol Phosphate Acyltransferase in Primary Monolayer Cultures of Parenchymal Cells from Adult Rat Liver

Previous studies have shown that isolation and primary culture of rat hepatocytes in a standard, chemically defined medium is associated with selective changes in microsomal function. These changes were found to be selectively sensitive to addition of hormones to the culture medium. The concentration of cytochrome P-450 declined dramatically during the first 24 hours of incubation. However, cytochrome P₁-450, a form of the hemoprotein induced by polycyclic aromatic hydrocarbons, was resistant to this change. Cytochrome P₁-450 levels selectively rose during the first ten hours in culture and, thereafter, declined at a less rapid rate than did the cytochrome P-450 in normal hepatocytes or in cells prepared from phenobarbital pretreated animals. Addition of dexamethasone to the medium at the time of cell plating partially prevented the fall of cytochrome P-450 and of ¹⁴C-heme in microsomes prepared from hepatocytes derived from rats given 5¹⁴[C]-δ-aminolevulinic acid. This suggests that the steroid decreases degradation of the hemoprotein. As compared to the loss of cytochrome P-450 in cultures of normal hepatocytes, the hemoprotein fell to lower levels in hepatocytes prepared from regenerated liver four days after partial hepatectomy. This result may be related to the accelerated formation of the monolayer in the cultures of regenerated hepatocytes. Both sn-glycerol-3-phosphate acyltransferase activity and glycerol kinase activity declined in the first 24 hours of culture. The fall in the latter enzyme was partially prevented by addition of estradiol. Collagen prolyl hydroxylase, a newly discovered microsomal constituent of the hepatocyte, rose slightly during the first 24 hours in culture. This change was augmented threefold by addition of insulin to the medium. We conclude that the present hepatocyte culture system with its attendant changes in functional phenotype may be useful in better defining the role of hormones in modulating metabolic processes in the liver.     

Pronounced and differential effects of ionic strength and pH on testosterone oxidation by membrane-bound and purified forms of rat liver microsomal cytochrome P-450

The aim of this study was to determine the effects of ionic strength and pH on the different pathways of testosterone oxidation catalyzed by rat liver microsomes. The catalytic activity of cytochromes P-450a (IIA1), P-450b (IIB1), P-450h (IIC11) and P-450p (IIIA1) was measured in liver microsomes from mature male rats and phenobarbital-treated rats as testosterone 7 alpha-, 16 beta-, 2 alpha- and 6 beta-hydroxylase activity, respectively. An increase in the concentration of potassium phosphate (from 25 to 250 mM) caused a marked decrease in the catalytic activity of cytochromes P-450a (to 8%), P-450b (to 22%) and P-450h (to 23%), but caused a pronounced increase in the catalytic activity of cytochrome P-450p (up to 4.2-fold). These effects were attributed to changes in ionic strength, because similar but less pronounced effects were observed with Tris-HCl (which has approximately 1/3 the ionic strength of phosphate buffer at pH 7.4). Testosterone oxidation by microsomal cytochromes P-450a, P-450b, P-450h and P-450p was also differentially affected by pH (over the range 6.8-8.0). The pH optima ranged from 7.1 (for P-450a and P-450h) to 8.0 (for P-450p), with an intermediate value of 7.4 for cytochrome P-450b. Increasing the pH from 6.8 to 8.0 unexpectedly altered the relative amounts of the 3 major metabolites produced by cytochrome P-450h. The decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h that accompanied an increase in ionic strength or pH could be duplicated in reconstitution systems containing purified P-450a, P-450b or P-450h, equimolar amounts of NADPH-cytochrome P-450 reductase and optimal amounts of dilauroylphosphatidylcholine. This result indicated that the decline in testosterone oxidation by cytochromes P-450a, P-450b and P-450h was a direct effect of ionic strength and pH on these enzymes, rather than a secondary effect related to the increase in testosterone oxidation by cytochrome P-450p. Similar studies with purified cytochrome P-450p were complicated by the atypical conditions needed to reconstitute this enzyme. However, studies on the conversion of digitoxin to digitoxigenin bisdigitoxoside by liver microsomes, which is catalyzed specifically by cytochrome P-450p, provided indirect evidence that the increase in catalytic activity of cytochrome P-450p was also a direct effect of ionic strength and pH on this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Chemical characterization of protein-protein interactions between cytochrome P-450 and cytochrome b5

Native cytochrome b5 interacts with either RLM5 or LM2 to form tight equimolar complexes (Kd = 250 and 540 nM, respectively) in which the content of high spin cytochrome P-450 was substantially increased. Cytochrome b5 caused 3- and 7-fold increases in the binding affinities of RLM5 and LM2 for benzphetamine, respectively, and benzphetamine decreased the apparent Kd for cytochrome b5 binding. Upon formation of the ternary complex between cytochromes P-450, b5, and benzphetamine the percentage of cytochrome P-450 in the high spin state was increased from 28 to 74 (RLM5) and from 9 to 85 (LM2). Cytochrome b5 caused 13- and 7-fold increases in the rate of RLM5- and LM2-dependent p-nitroanisole demethylation, respectively. Amino-modified (ethyl acetimidate or acetic anhydride) cytochrome b5 produced results similar to those obtained above with native cytochrome b5. In contrast, modification of as few as 5 mol of carboxyl groups/mol of amidinated cytochrome b5 resulted in both a substantial loss of the spectrally observed interactions with either cytochrome P-450 LM2 or cytochrome P-450 RLM5, and in a loss of the cytochrome b5-mediated stimulation of p-nitroanisole demethylation catalyzed by either monooxygenase. In further studies, native and fully acetylated cytochromes b5 reoxidized carbonmonoxy ferrous LM2 at least 20 times faster than amidinated, carboxyl-modified cytochrome b5 derivatives. In contrast, amidination, or acetylation of amino groups, or amidination of amino groups plus methylamidination of the carboxyl groups did not appreciably slow the rate of reduction of the cytochrome b5 by NADPH-cytochrome P-450 reductase. Collectively, the results provide strong evidence for an essential role of cytochrome b5 carboxyl groups in functional interactions with RLM5 and LM2.     

Maintenance and induction of cytochrome P-450 in cultured rat hepatocytes

Maintenance of microsomal cytochrome P-450 content by cultured rat hepatocytes has proven an elusive goal. It is reported here that exogenous heme maintains cytochrome P-450 content of cultured rat hepatocytes at high levels during the first 72 h of incubation. The maintenance studies have been expanded to demonstrate the in vitro induction of cytochrome P-450 by phenobarbital treatment. The induction of P-450 in vitro by phenobarbital required the trace element, selenium, in the presence of exogenous heme. The present findings suggest that selenium, and other trace elements, may have an essential role in the formation of holocytochrome P-450 in vitro.     

Induction of cytochrome P-450 and ethanol oxidation in Yarrowia lipolytica

The study of the effect of different ethanol concentrations in the medium on the growth and the activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. But when the initial concentration of ethanol in the medium was 3 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Regioselective progesterone hydroxylation catalyzed by eleven rat hepatic cytochrome P-450 isozymes

Quantitative high-pressure liquid chromatographic assays were developed that separate progesterone and 17 authentic monohydroxylated derivatives. The assays were utilized to investigate the hydroxylation of progesterone by 11 purified rat hepatic cytochrome P-450 isozymes and 8 different rat hepatic microsomal preparations. In a reconstituted system, progesterone was most efficiently metabolized by cytochrome P-450h followed by P-450g and P-450b. Seven different monohydroxylated progesterone metabolites were identified. 16 alpha-Hydroxyprogesterone, formed by 8 of the 11 isozymes, was the only detectable metabolite formed by cytochromes P-450b and P-450e. 2 alpha-Hydroxyprogesterone was formed almost exclusively by cytochrome P-450h, and 6 alpha-hydroxyprogesterone and 7 alpha-hydroxyprogesterone were only formed by P-450a. 6 beta-hydroxylation of progesterone was catalyzed by four isozymes with cytochrome P-450g being the most efficient, and 15 alpha-hydroxyprogesterone was formed as a minor metabolite by cytochromes P-450g, P-450h, and P-450i. None of the isozymes catalyzed 17 alpha-hydroxylation of progesterone, and only cytochrome P-450k had detectable 21-hydroxylase activity. 16 alpha-Hydroxylation catalyzed by cytochrome P-450b was inhibited in the presence of dilauroylphosphatidylcholine (1.6-80 microM), while this phospholipid either stimulated (up to 3-fold) or had no effect on the metabolism of progesterone by the other purified isozymes. Results of microsomal metabolism in conjunction with antibody inhibition experiments indicated that cytochromes P-450a and P-450h were the sole 7 alpha- and 2 alpha-hydroxylases, respectively, and that P-450k or an immunochemically related isozyme contributed greater than 80% of the 21-hydroxylase activity observed in microsomes from phenobarbital-induced rats.     

Inhibition by cycloheximide of degradation of cytochrome P-450 in primary cultures of adult rat liver parenchymal cells and in vivo

Degradation of cytochrome P-450 was studied in adult rat liver parenchymal cells in primary monolayer culture. In cells incubated in standard culture medium, the amount of cytochrome P-450 decreased at an accelerated rate relative to either the rate of degradation of total protein in the cells or the turnover of cytochrome P-450 in vivo. This change was succeeded by a spontaneous increase in the activity of haem oxygenase, an enzyme system that converts haem into bilirubin in vitro, measured in extracts from the cultured cells. This finding suggests that the rate of cytochrome P-450 breakdown may be controlled by factor(s) other than the activity of haem oxygenase. The decline in cytochrome P-450 and the subsequent increase in haem oxygenase activity was prevented by incubation of hepatocytes in medium containing an inhibitor of protein synthesis such as cycloheximide, puromycin, actinomycin D, or azaserine. The effect of cycloheximide appeared to be due to decreased breakdown of microsomal (14)C-labelled haem. By contrast, cycloheximide was without effect on the degradation of total protein, measured either in homogenates or in microsomal fractions prepared from the cultured cells. These results suggest that the conditions of cell culture stimulate selective degradation of cytochrome P-450 by a process that is inhibited by cycloheximide and hence may require protein synthesis. The findings in culture were verified in parallel studies of cytochrome P-450 degradation in vivo. After administration of bromobenzene, the degradation of the haem moiety of cytochrome P-450 was accelerated in vivo in a manner resembling that observed in cultured hepatocytes. Administration of cycloheximide to either bromobenzene-treated rats or to untreated rats decreased the degradation of the haem moiety of cytochrome P-450. However, the drug failed to affect degradation of haem not associated with cytochrome P-450, suggesting that cycloheximide is not a general inhibitor of haem oxidation in the liver. These findings confirm that the catabolism of hepatic cytochrome P-450 haem is controlled by similar cycloheximide-sensitive processes in the basal steady state in vivo, as stimulated by bromobenzene in vivo, or in hepatocytes under the conditions of cell culture. We conclude that the rate-limiting step in this process appears to require protein synthesis and precedes cleavage of the haem ring.     

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.     

Regulation of steroidogenesis in rat Leydig cells in culture: effect of human chorionic gonadotropin and dibutyryl cyclic AMP on the synthesis of cholesterol side chain cleavage cytochrome P-450 and adrenodoxin

Rat Leydig cells in primary culture were used as a model system to investigate the effects of human chorionic gonadotropin (hCG) and dibutyryl cyclic AMP (Bt2cAMP) on the synthesis of cholesterol side chain cleavage cytochrome P-450 (cytochrome P-450scc) and the iron-sulfur protein, adrenodoxin. Leydig cells isolated from the testes of mature rats were placed in monolayer culture in the absence of stimulatory factors for 8 days. HCG (10 mIU/ml) or Bt2cAMP (1 mM) were then added to some of the cultures and the incubations were continued for up to 48 h. Testosterone production was increased markedly in cells incubated with hCG or Bt2cAMP. A significant accumulation of pregnenolone in the medium of cells treated with Bt2cAMP was also observed. Both hCG and Bt2cAMP increased the rates of synthesis of cytochrome P-450scc and adrenodoxin. In hCG-treated cells the apparent rate of synthesis of cytochrome P-450scc was increased 13-fold over that of controls after 48 h of incubation; the rate of adrenodoxin synthesis was increased 4-fold by hCG treatment. In Bt2cAMP-treated cells the rate of synthesis of cytochrome P-450scc was 37-fold greater than that of control cells after 48 h of incubation; adrenodoxin synthesis was increased 36-fold over controls. In hCG- and Bt2cAMP-treated cells, the concentration of immunoreactive cytochrome P-450scc and adrenodoxin increased with increasing time of incubation, and were correlated with the stimulatory effects of these agents on cytochrome P-450scc activity and on total steroid production. The results of this study are indicative that the maintenance by LH/hCG of elevated levels of testosterone synthesis by the Leydig cell is mediated, in part, by induction of the synthesis of cytochrome P-450scc and its associated protein, adrenodoxin. Since Bt2cAMP had effects similar to those observed with hCG, it is suggested that the stimulatory effects of hCG on the synthesis of cytochrome P-450scc and adrenodoxin are mediated by increased cyclic AMP formation.     

Properties of a soluble inositol 1,3,4,5-tetrakisphosphate 3-phosphatase from porcine brain.

Polyclonal antibodies to the major beta-naphthoflavone (BNF)-inducible form of cytochrome P-450 (P450IA) and to the major phenobarbitone (PB)-inducible form (P450IIB) have been used to quantify the contribution of these subfamilies to the total amount of cytochrome P-450 in rat livers and rat hepatocyte cultures treated with PB, BNF and metyrapone for 24 and 72 h. The P450IA and IIB subfamilies were not detectable (less than 5 pmol/mg of microsomal protein) in the livers of control rats, but administration of BNF resulted in the P450IA subfamily comprising more than 80% of the total hepatic cytochrome P-450. Administration of PB and metyrapone to rats did not elevate the level of this subfamily but elevated the levels of the P450IIB subfamily to 60% and 30% respectively of the total. Thus metyrapone is a ''PB-like'' inducer. However, in contrast with their effects in vivo, treatment with PB and metyrapone of rat hepatocytes did not elevate the proportion of the P450IIB subfamily relative to that in untreated cells but rather, like BNF, increased the P450IA subfamily. This would account for the ability of metyrapone to produce in hepatocyte culture, like BNF, a pronounced induction of ethoxyresorufin O-de-ethylase activity, but it does not account for why of all inducers studied only metyrapone can maintain the total cytochrome P-450 content of cultured hepatocytes, or the activity of ethylmorphine N-demethylase. This activity is generally considered to be associated with the P450IIB subfamily, but the lack of effect of metyrapone on this subfamily in hepatocyte culture must suggest that metyrapone is able to prevent the loss of the total amount of the cytochrome by increasing the expression of other cytochromes P-450.     

The Induction of Cytochrome P-450 and Ethanol Oxidation in Yarrowia lipolytica Cells

The study of the effect of different ethanol concentrations in the medium on the growth and activity of enzymatic systems involved in ethanol oxidation in Yarrowia lipolytica showed that the cultivation of yeast cells on 1 and 2% ethanol caused their rapid growth and a drastic increase in cell respiration and sensitivity to cyanide already in the first hours of cultivation. At the same time, during cultivation on 3, 4, and 5% ethanol, the growth and respiration of yeast cells were considerably suppressed. All of the ethanol concentrations studied induced the synthesis of cytochrome P-450, its dynamics in cells being dependent on the initial concentration of ethanol in the medium. When the initial concentration of ethanol was 1 and 2%, the content of cytochrome P-450 in cells steeply decreased after a short period of induction. However, when the initial concentration of ethanol in the medium was 4 to 5%, the content of cytochrome P-450 in cells was high throughout the cultivation period. The induction of cytochrome P-450 in cells preceded the induction of the NAD-dependent enzymes alcohol dehydrogenase and catalase, which, like cytochrome P-450, are also involved in ethanol oxidation by yeasts. The activity of catalase was higher in the yeast cells grown in the presence of 3 to 5% ethanol than in the cells grown in the presence of 1 and 2% ethanol. The roles played by cytochrome P-450, alcohol dehydrogenase, and catalase in ethanol oxidation by yeast cells are discussed.     

Thermodynamic studies of the protein-protein interactions between cytochrome P-450 and cytochrome b5. Evidence for a central role of the cytochrome P-450 spin state in the coupling of substrate and cytochrome b5 binding to the terminal hemoprotein

The interactions between purified rat hepatic microsomal cytochrome P-450 and the type I ligands benzphetamine and cytochrome b5 have been studied in the presence of phospholipid using difference spectrophotometry. Cytochrome b5 was shown to interact with cytochrome P-450 to form a tight 1:1 complex (Kd = 275 nM), in which the proportion of high spin cytochrome P-450 was increased from 7 to 30%. The presence of saturating cytochrome b5 was shown to cause a decrease in the apparent Kd for benzphetamine binding from 111 microM to 40 microM. Likewise, the presence of benzphetamine was shown to cause a decrease in the apparent dissociation constant for cytochrome b5 binding to cytochrome P-450 (Kd = 90 nM). The above interactions were resolved into the basic equilibria inter-relating the various ligation states of the hemoprotein in an energetically closed eight-state free energy coupling model and the relative magnitudes of the microequilibria were analyzed to determine the degree of coupling of the interactions between cytochrome P-450 and both benzphetamine and cytochrome b5. Consequently, the spin state changes in cytochrome P-450 induced by benzphetamine and cytochrome b5 binding were shown to arise because these ligands interact 7 and 4 times more tightly with high spin cytochrome P-450, respectively. Furthermore, the data revealed that these ligands interact at independent sites on cytochrome P-450. Thus the effects of cytochrome b5 upon benzphetamine binding and vice versa were rationalized simply in terms of an increase in the proportion of a high spin (high affinity) conformation of cytochrome P-450 brought about by pre-equilibration with the effector ligand, with the intrinsic binding affinities of the two ligands for the low or high spin states remaining relatively unaltered. The thermodynamic parameters associated with the interactions between cytochrome P-450 and cytochrome b5, determined from the temperature dependence of these interactions, revealed that these protein interactions are entropy driven and probably occur by a hydrophobic mechanism.