Hepatic P-450 cholesterol 7 alpha-hydroxylase. Regulation in vivo at the protein and mRNA level in response to mevalonate, diurnal rhythm, and bile acid feedback

Cholesterol 7 alpha-hydroxylase (P-450 Ch7 alpha) catalyzes the first and rate-limiting step in the hepatic conversion of cholesterol to bile acids. P-450 Ch7 alpha activity in rat liver is regulated at three independent levels: (a) feedback inhibition by bile acids (long term regulation); (b) midterm regulation through the diurnal cycle; (c) short term modulation by hormones and dietary factors. P-450 Ch7 alpha was purified to apparent homogeneity and in active form (turnover number = 10-15 min-1 P-450(-1)) from cholestyramine-fed female rats, and rabbit anti-P-450 Ch7 alpha polyclonal antibodies were then prepared. Liver microsomes were isolated from rats fed normal diet or diet containing the bile acid sequestrant cholestyramine and were then killed at either the apex (midnight) or nadir (noon) of the diurnal rhythm of P-450 Ch7 alpha activity. Direct comparison of microsomal P-450 Ch7 alpha enzyme activity levels with P-450 Ch7 alpha protein (Western blotting) and mRNA levels (Northern and slot blots) revealed that the 2.5-3-fold induction of P-450 Ch7 alpha activity with cholestyramine feeding can be fully accounted for by an increase in P-450 Ch7 alpha protein and mRNA. Turnover numbers of 7-9 nmol of 7 alpha-hydroxycholesterol/min/nmol of microsomal P-450 Ch7 alpha were observed for both induced and uninduced animals. Similarly, the postmidnight decrease in enzyme activity could be generally accounted for by a decrease in P-450 Ch7 alpha protein and mRNA, suggesting that these species have relatively short half-lives. The short term regulation of P-450 Ch7 alpha was examined following treatment with the cholesterol precursor mevalonic acid. A 2.5-fold increase in hepatic microsomal P-450 Ch7 alpha activity occurred within 150 min and was accompanied by a significant elevation of P-450 Ch7 alpha mRNA (up to 3-6-fold increase). These findings establish that hepatic cholesterol 7 alpha-hydroxylase activity is regulated in response to long term, midterm, and short term control factors primarily at a pretranslational level and that this regulation is of greater importance than proposed mechanisms based on allosteric effects of bile acids on P-450 Ch7 alpha protein, changes in cholesterol availability, or reversible phosphorylation of a putative P-450 Ch7 alpha phosphoprotein.     

Cytochrome P-450 cholesterol 7 alpha-hydroxylase: inhibition of enzyme deactivation by structurally diverse calmodulin antagonists and phosphatase inhibitors

Cytochrome P-450 cholesterol 7 alpha-hydroxylase (P-450Ch7 alpha) catalyzes the first and rate-limiting step in the conversion of cholesterol to bile acids. Incubation of rat liver microsomes in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer resulted in a time-dependent deactivation of P-450Ch7 alpha which was markedly accelerated by the nonionic detergent Tween 80. Microsomal NADPH-cytochrome P-450 reductase and cytochrome P-450-dependent 7-ethoxycoumarin O-deethylase activities were unaffected under these conditions, evidencing the selectivity of the deactivation process for P-450Ch7 alpha. The rate (t 1/2 = 15-19 min at 37 degrees C) and maximal extent of P-450Ch7 alpha deactivation (greater than or equal to 90%) were both unaffected by the presence of cytosolic proteins and were also not dependent on the initial enzyme level, as shown using liver microsomes isolated from untreated, cholestyramine-fed, and xenobiotic-induced rats exhibiting an eight-fold range in P-450Ch7 alpha activity. Scavengers for reduced oxygen species were also without effect. P-450Ch7 alpha was stabilized some six- to sevenfold (t 1/2 = 94-143 min) by the phosphatase inhibitor NaF. Of a series of other phosphatase inhibitors examined, including, among others, EDTA, vanadate, and molybdate, only phosphate-containing compounds and the calmodulin antagonist trifluoperazine, and inhibitor of the Ca2+-calmodulin-dependent phosphatase calcineurin, effectively stabilized P-450Ch7 alpha. Modulation of P-450Ch7 alpha deactivation by these inhibitors generally paralleled their effects on isolated calcineurin. A variety of structurally diverse calmodulin antagonists examined were also found to effectively protect P-450Ch7 alpha from deactivation; these include calmidazolium and tamoxifen (IC50 = 25 to 50 microM), chlorpromazine, thioridazine, amitriptyline, imipramine, and the naphthalene sulfonamide compound W-7 (IC50 = 50 to 300 microM). Structure-activity analysis of several phenothiazines and their derivatives indicated that although little activity was exhibited by the sulfoxides, some protection was provided by the corresponding sulfones. On the basis of these observations, various models for the molecular basis of enzyme deactivation are considered, including the hypothesis that a calcineurin-like microsomal phosphatase mediates deactivation of this cytochrome P-450 enzyme.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Inhibition of cholesterol 7 alpha-hydroxylase by an antibody to a male-specific form of cytochrome P-450 from subfamily P450IIC.

The absence of antibodies to cholesterol 7 alpha-hydroxylase (EC 1.14.13.17), the rate-determining enzyme for bile acid synthesis, has significantly compromised studies on this protein. Nine antibodies raised against proteins from the cytochrome P-450 gene families (P450I, P450IIA, P450IIB, P450IIC and P450III) were tested as inhibitors of 7 alpha-hydroxylase activity. An antibody raised against a male-predominant P-450 (PB2a, P450h) from the P450IIC gene subfamily was an effective inhibitor of activity in liver microsomal fractions from rat, mouse and hamster. The inhibition could be reversed by the addition of PB2a antigen, indicating structural similarity between cholesterol 7 alpha-hydroxylase and proteins within the P450IIC subfamily. Western blot analysis of hepatic microsomal fractions with the PB2a antibody gave three bands, two of which, like cholesterol 7 alpha-hydroxylase, did not inhibit sexual dimorphism. The intensity of one of the bands (apparent Mr 54,000) correlated with changes observed in activity due to diet [Spearman correlation of 0.800 (P less than 0.01)]. These findings suggest that cholesterol 7 alpha-hydroxylase is a form of P-450 which shares structural similarity with cytochromes P-450 in the P450IIC gene subfamily and that its feedback regulation by bile acid involves protein induction rather than simply post-translational modification.     

Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme

Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.     

Purification of cholesterol 7 alpha-hydroxylase from human and rat liver and production of inhibiting polyclonal antibodies

Cholesterol 7 alpha-hydroxylase, the cytochrome P-450-dependent and rate-controlling enzyme of bile acid synthesis, was purified from rat and human liver microsomes. The purified fractions were assayed in a reconstituted system containing [4-14C]cholesterol, and cholesterol 7 alpha-hydroxylase activities in these fractions increased 500-600-fold relative to whole microsomes. Polyacrylamide gel electrophoresis of rat microsomes followed by immunoblotting with polyclonal rabbit antisera raised against purified cholesterol 7 alpha-hydroxylases revealed two peaks at molecular masses of 47,000 and 49,000 daltons for both rat and human fractions. Increasing amounts of rabbit anti-rat and anti-human antibodies progressively inhibited rat microsomal cholesterol 7 alpha-hydroxylase activity up to 80%. In contrast, monospecific antibodies raised against other purified cytochrome P-450 enzymes (P-450f, P-450g, and P-450j) did not inhibit rat or human cholesterol 7 alpha-hydroxylase activity. Immunoblots of rat microsomes with the rabbit anti-rat cholesterol 7 alpha-hydroxylase antibody demonstrated that the antibody reacted quantitatively with the rat microsomal enzyme. Microsomes from cholesterol-fed rats showed increased cholesterol 7 alpha-hydroxylase mass, whereas treatment with pravastatin, an inhibitor of hydroxy-methylglutaryl-coenzyme A reductase, reduced enzyme mass. Microsomes from starved rats contained slightly less cholesterol 7 alpha-hydroxylase protein than chow-fed control rats. These results indicate a similarity in molecular mass, structure, and antigenicity between rat and human cholesterol 7 alpha-hydroxylases; demonstrate the production of inhibiting anti-cholesterol 7 alpha-hydroxylase antibodies that can be used to measure the change in cholesterol 7 alpha-hydroxylase enzyme mass under various conditions; and emphasize the unique structure of cholesterol 7 alpha-hydroxylase with respect to other cytochrome P-450-dependent hydroxylases.     

Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression

Rat hepatic cytochrome P-450 form RLM2 is a testosterone 15 alpha-hydroxylase reported to be male-specific on the basis of purification studies (Jansson, I., Mole, J., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 7084-7093). The sex dependence, developmental regulation, xenobiotic induction, and hormonal control of P-450 RLM2 expression were studied using P-450 form-specific immunochemical and catalytic assays. Polyclonal antibodies raised to rat hepatic P-450 3 (P-450 gene IIA1) were found to cross-react strongly with P-450 RLM2, but not with 10 other rat P-450 forms, suggesting that P-450 3 and P-450 RLM2 are highly conserved in primary structure. Western blotting of liver microsomes under conditions where P-450s 3 and RLM2 are resolved electrophoretically revealed that P-450 RLM2 is markedly induced at puberty in male rats, with no protein detected (less than or equal to 5% of adult male levels) in adult females or immature animals of either sex. A similar developmental dependence was observed for hepatic microsomal testosterone 15 alpha-hydroxylase activity, which was found to be catalyzed primarily by P-450 RLM2. P-450 RLM2 was resistant to induction by several xenobiotics and in the case of phenobarbital and beta-naphthoflavone, was suppressed by 50-60%. Studies on the steroid hormonal regulation of P-450 RLM2 revealed that its adult male-specific expression is imprinted (programmed) in response to neonatal testosterone exposure. Ovariectomy studies demonstrated that suppression by estrogen does not contribute significantly to the absence of P-450 RLM2 in adult female rats. Although the male-specific developmental induction of P-450 RLM2 in response to neonatal testosterone is strikingly similar to that of P-450 2c (testosterone 2 alpha/16 alpha-hydroxylase; gene IIC11), P-450 RLM2 expression is not dependent on the pulsatile pituitary growth hormone secretion required for P-450 2c synthesis. Rather, hypophysectomy of adult male rats increased P-450 RLM2 and its associated testosterone 15 alpha-hydroxylase activity by 50-100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of cytochrome P-450 in cholesterol biogenesis and catabolism

1. Adjuvant-induced arthritis in rats is accompanied by a loss of activity of the drug-metabolizing enzyme system and a decrease in hepatic cytochrome P-450. 2. Arthritic rats have normal serum and liver cholesterol concentrations. 3. The rate of biogenesis of cholesterol in vivo and in vitro from either [(14)C]acetate or [(14)C]mevalonate in arthritic rats was the same as or greater than that found in control rats. 4. Treatment of rats with carbon disulphide (1ml/kg) resulted in a loss of drug-metabolizing-enzyme activity and increased cholesterol biogenesis. 5. The activity of cholesterol 7alpha-hydroxylase in adjuvant-induced arthritic rats did not differ significantly from that in control rats. 6. Rats fed with cholestyramine had an elevated hepatic cholesterol 7alpha-hydroxylase activity, but neither the concentration of cytochrome P-450 nor the activity of the drug-hydroxylating enzyme, aminopyrine demethylase, was affected. 7. The relationships between drug hydroxylation and cholesterol metabolism are discussed.     

Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cytochrome P-450 apoprotein in the rat

The hormonal regulation of the sexually differentiated cytochrome P-450 isozyme which catalyzes 16 alpha-hydroxylation of testosterone and 4-androstene-3,17-dione in male rat liver (P-450(16) alpha) was investigated. Estradiol valerate injection of male rats caused a decrease in P-450(16) alpha levels to almost the female level, while methyltrienolone injection had the reverse effect in female animals. Hypophysectomy abolished the sex difference in P-450(16) alpha levels. Human growth hormone infusion into male rats, mimicking the female pattern of growth hormone secretion, caused a feminization of P-450(16) alpha levels. The same effect was also seen in hypophysectomized rats of both sexes. In contrast, a different administration schedule involving 12 h injections of human growth hormone, mimicking the male pattern of growth hormone secretion, caused a masculinization of P-450(16) alpha levels in hypophysectomized rats, at a daily dose which causes feminization when given by infusion. Thus, the level of expression of P-450(16) alpha in the liver is dependent on the temporal pattern of blood growth hormone levels. While infusion of rat growth hormone into male rats also feminized the P-450(16) alpha levels, infusion of ovine prolactin had no effect. Ontogenic studies showed that the developmental pattern of P-450(16) alpha expression in the liver coincided with the known pattern of development of the sexual differentiation of hepatic steroid 16 alpha-hydroxylase activity and of the diurnal pattern of growth hormone secretion.     

Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7 alpha-hydroxylase

A complete cDNA clone encoding human cholesterol 7 alpha-hydroxylase has been isolated using a rat P-450ch7 alpha cDNA insert [(1989) FEBS Lett. 257, 97-100] as a probe and totally sequenced. The cDNA contained 1512-base pair open reading frame encoding 504 amino acid residues (Mr 57,630), 39-base pair 5'-untranslated region 1322-base pair 3'-ultranslated region including 20 nucleotides of poly A tail in the total length of 2873 base pairs. The deduced amino acid sequence showed 82% similarity to rat P-450ch7 alpha. Unique amino acid residues were observed in putative binding domains for heme and steroid which are highly conserved in most steroidogenic P-450s.     

Genetic regulation of testosterone 15 alpha-hydroxylase (cytochrome P-450(15)alpha) in renal microsomes of female mice

P-450(15)alpha is a form of cytochrome P-450 purified from liver microsomes of female 129/J mice that is specific for oxidation of testosterone to its 15 alpha-hydroxylated product. Testosterone 15 alpha-hydroxylase activity that was inhibited by anti-P-450(15)alpha antibody was approximately 50 times higher in renal microsomes from 129/J than in BALB/cJ females. Western blots of renal microsomes using anti-P-450(15)alpha antibody showed the presence of immunoreactive protein with a molecular weight identical with that of hepatic P-450(15)alpha in 129/J but not in BALB/cJ female mice. To investigate the genetic basis for the strain differences in this activity, the distribution of P-450(15)alpha-dependent testosterone 15 alpha-hydroxylase activity in renal microsomes from individual females of 129/J and BALB/cJ, of F1 offspring of these strains, and of F1 back-crosses to the progenitor strains were determined. The results were consistent with a sex-related autosomal dominant regulation of the higher activity in 129/J females by a single locus, designated Rsh (regulation of steroid hydroxylase). The amounts of immunochemically cross-reactive P-450(15)alpha protein were linearly correlated with testosterone 15 alpha-hydroxylase activities in renal microsomes from Rsh heterozygotes and homozygotes. At least twice as much mRNA, which hybridized with the cDNA clone for hepatic P-450(15)alpha, was detected in 129/J and 129CF1/J compared to BALB/cJ female kidneys. The evidence suggests a pretranslational regulation of the P-450(15)alpha isozyme in the female mouse kidney by the Rsh locus.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of cholesterol 7 alpha-hydroxylase from rat liver microsomes

Cholesterol 7 alpha-hydroxylase (cholesterol, NADPH: oxygen oxidoreductase, 7 alpha-hydroxylating, EC 1.14.13.17) was purified from liver microsomes of cholestryramine-fed male rats by using high-performance ion-exchange chromatography. The purified enzyme showed a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr = 52,000), and its dithionite-reduced CO complex exhibited an absorption maximum at 450 nm. The specific content of the enzyme was 9 nmol of cytochrome P-450/mg of protein. Upon reconstitution with NADPH-cytochrome P-450 reductase, the enzyme showed a high activity of cholesterol 7 alpha-hydroxylation with the turnover number of 50 min-1 at 37 degrees C. The reaction was inhibited neither by aminoglutethimide nor by metyrapone, but inhibited markedly by iodoacetamide and disulfiram. The reaction was also inhibited significantly by CO. The enzyme catalyzed hydroxylation of cholesterol with strict regio- and stereoselectivity and was inert toward other sterols which are intermediates in the conversion of cholesterol to bile acids, i.e. 7 alpha-hydroxy-4-cholesten-3-one (12 alpha-hydroxylation), 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol (25-hydroxylation), and taurodeoxycholate (7 alpha-hydroxylation). Unlike other cytochromes P-450 isolated from rat liver microsomes, the enzyme showed no activity toward testosterone and xenobiotics such as 7-ethoxycoumarin and benzo[a] pyrene. The NH2-terminal amino acid sequence of the enzyme was Met-Phe-Glu-Val(Ile)-Ser-Leu-, which was distinct from those of any other cytochromes P-450 of rat liver microsomes hitherto reported. These results indicate that the enzyme is a novel species of cytochrome P-450 so far not isolated from liver microsomes.     

Mouse steroid 15 alpha-hydroxylase gene family: identification of type II P-450(15)alpha as coumarin 7-hydroxylase

We identified type II P-450(15)alpha as mouse coumarin 7-hydroxylase (P-450coh). Unlike type I P-450(15)alpha, the other member within the mouse steroid 15 alpha-hydroxylase gene family, type II catalyzed little steroid 15 alpha-hydroxylase activity, yet structurally there were only 11 substitutions between type I and type II P-450(15)alphaS within their 494 amino acid residues (Lindberg et al., 1989), and the N-terminal sequence (21 residues) of P-450coh was identical with that of both P-450(15)alphaS. Induction by pyrazole of coumarin 7-hydroxylase activity correlated well with the increase of type II P-450(15)alpha mRNA in 129/J male and female mice. Pyrazole, on the other hand, was less in males or not effective in females in inducing the 15 alpha-hydroxylase activity and type I P-450(15)alpha mRNA. Expression of type I and II in COS-1 cells revealed that the latter catalyzed coumarin 7-hydroxylase activity at 10 to approximately 14 pmol min-1 (mg of cellular protein)-1. The former, on the other hand, had a high testosterone 15 alpha-hydroxylase but little coumarin 7-hydroxylase activity. It was concluded, therefore, that type II P-450(15)alpha is the mouse coumarin 7-hydroxylase. Identification of type II as the P-450 specific to coumarin 7-hydroxylase activity and characterization of its cDNA and gene, therefore, were significant advances toward understanding the basis of genetic regulation of this activity in mice (known as Coh locus).     

Cholesterol 7 alpha-hydroxylase in isolated rat liver cells.

1. The activity of cholesterol 7 alpha-hydroxylase found in the 10000 x g supernatant prepared from isolated rat liver cells was comparable to that found with microsomal fractions from whole liver. 2. The activity of cholesterol 7 alpha-hydroxylase from cells prepared from livers of rats fed the bile salt sequestering agent cholestyramine was 2--3 fold higher than the activity of this enzyme found in cells isolated from animals on a control diet. 3. On incubation of hepatocytes in a suitable medium at 37 degrees C, cholesterol 7 alpha-hydroxylase activity declined to about 50% of its original value after three hours despite the fact that the cells retained a high level of viability over 5--6 h as measured by various sensitive criteria. 4. The decrease in cholesterol 7 alpha-hydroxylase activity was observed whether cholestyramine was included in the diet or excluded from the diet of the animals used as sources of the liver cells. 5. The change in cholesterol 7 alpha-hydroxylase activity seen on incubation of the cells was not affected by including in the incubation medium additional nutrients such as amino acids, the glucocorticoid cortisol, phospholipid dispersions, or sodium taurocholate. 6. Changing the incubation medium in which the cells were suspended at regular intervals during the three-hour experiments failed to prevent this decline in the cholesterol 7 alpha-hydroxylase activity during the incubation of these cells. 7. Although isolated liver cells have been shown to lose glutathione on incubation, addition of physiological levels of this compound did not prevent the decline in cholesterol 7 alpha-hydroxylase activity. 8. Cycloheximide addition to the incubation medium accelerated the decrease in cholesterol 7 alpha-hydroxylase activity. This suggests that some protein synthesis associated with cholesterol 7 alpha-hydroxylase activity occurs during the incubation and inhibition of such protein synthesis accelerated the decrease in this enzyme activity. 9. The cytochrome P-450 content of the 10000 x g supernatant prepared from hepatocytes declined slowly to about 65% of its original value after four hours of incubation at 37 degrees C. This decline in the 10000 x g supernatant cytochrome P-450 content may partly explain the observed loss of cholesterol 7 alpha-hydroxylase activity during incubations in vitro. 10. Isolated hepatocytes rapidly take up radioactively labelled sodium cholate. Subsequent excretion of the radioactivity was also very rapid even in the presence of large amounts of this bile salt in the medium.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Purification of cholesterol 7 alpha-hydroxylase and the immunochemical evidence for the induction of cholesterol 7 alpha-hydroxylase by cholestyramine and circadian rhythm

Two cholesterol 7 alpha-hydroxylase isozymes were purified from liver microsomes of cholestyramine-treated female rats by using anion exchange high performance liquid chromatography. These two cytochrome P-450 isozymes were similar in electrophoretic mobility, immunocross-reactivity, and Vmax but differed in Km for cholesterol, turnover number, and charges. Antibody against the major isozyme was raised in rabbit. This antibody specifically inhibited microsomal cholesterol 7 alpha-hydroxylase activity. Immunoblot of microsomal polypeptides indicated that microsomal cholesterol 7 alpha-hydroxylase enzyme levels were increased in parallel with cholesterol 7 alpha-hydroxylase activity upon the treatment of rats with diet supplemented with cholestyramine. Both cholesterol 7 alpha-hydroxylase activity and enzyme levels were drastically reduced immediately after the removal of cholestyramine from the diet. Cholesterol 7 alpha-hydroxylase activity was also detected in the microsomes of kidney, heart, and lung in about 7-27% of the level found in the liver. 3-Methylcholanthrene treatment induced cholesterol 7 alpha-hydroxylase activity and enzyme level. In contrast, pregnenolone-16 alpha-carbonitrile or dexamethasone treatment greatly depressed enzyme and activity in rats. Cholesterol 7 alpha-hydroxylase enzyme level was 2-3-fold higher in liver microsomes of rats maintained under the reversed light cycle than under the normal light cycle. In genetically obese Zucker rats, cholesterol 7 alpha-hydroxylase activity and enzyme level did not respond to the change in the light cycle, however, were induced to the same levels as in the lean rats by cholestyramine treatment. This study provided the first direct evidence that the bile acid feedback regulation and circadian rhythm of microsomal cholesterol 7 alpha-hydroxylase activity involved the induction of cholesterol 7 alpha-hydroxylase enzyme level.     

Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.     

Structure of the rat gene encoding cholesterol 7 alpha-hydroxylase

Cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase) is a microsomal cytochrome P-450 that catalyzes the first and rate-limiting step in bile acid biosynthesis, the major catabolic pathway in cholesterol homeostasis. The gene encoding the rat 7 alpha-hydroxylase has been isolated and characterized. Southern blotting experiments demonstrated that the gene is present in a single copy in the rat genome. DNA sequence analysis showed that the 7 alpha-hydroxylase gene is unique among the characterized cytochrome P-450s in that it contains only six exons. Nuclease S1 and primer-extension mapping experiments positioned the 5'-ends of the 7 alpha-hydroxylase mRNA approximately 20-25 nucleotides downstream of a consensus TATAAA sequence. RNA blotting experiments demonstrated the presence of multiple 7 alpha-hydroxylase mRNAs that differ in the lengths of their 3'-untranslated regions.