CHAPTER 38

Cholesterol 7 a-Hydroxylase and 12a-Hydroxylase

K. OKUDA, T. OGISHIMA, and M. NOSHIRO

A. Introduction Three major cytochrome P450 enzymes are involved in the conversion of cholesterol into bile acids, i.e., cholesterol 7a-hydroxylase (EC 1.14.13.17), 12a-hydroxylase and 5~-cholestane-3a,7a,12a-triol 27-hydroxylase (EC 1.14.13.15) of which cholesterol 7a-hydroxylase catalyzes a rate-limiting step for overall conversion, whereas 12a-hydroxylase seems to regulate the relative amount of cholic acid over chenodeoxycholic acid (BJORKHEM 1985). 27-Hydroxylase catalyzes the hydroxylation of one of the terminal methyl groups of 5~-cholestane-3a,7a,12a-triol and 5~-cholestane-3a,7a-diol. In this chapter cholesterol 7u-hydroxylase and 12a-hydroxylase are reviewed. These two hydroxylases modify the steroid nucleus (Fig. 1). The cytochrome P450 species involved in side chain oxidation is reviewed elsewhere in this book by WIKVALL (see Chap. 46).

B. Cholesterol 7(1-Hydroxylase (CYP7)

I. Physiological Significance

Cholesterol 7a-hydroxylase (cytochrome P450ch7a) is localized in the endo­plasmic reticulum of hepatic parenchmal cells and its activity is regulated at three levels, i.e., long-term, mid-term and short-term regulations (MYANT and MITROPOULOS 1977). In long-term regulation, the bile acid biosynthesis is controlled by a feedback inhibition mechanism in which bile salts are reabsorbed in the ileum and then brought to the liver where they inhibit their own production via the enterohepatic circulation system. Bile acid binding resins added to the diet, e.g., cholestyramine, disrupt this feedback loop, elevating cholesterol 7a-hydroxylase activity and bile acid production (SHEFER et al. 1969; RHEINER et al. 1989). This increase in enzyme activity requires several days to be established fully, indicating that the negative feedback control by bile salts is a form of long-term regulation. In mid­term regulation the enzyme activity reveals a diurnal rhythm, with enzyme activity high at night and low during the day in the rat (GIELEN et al. 1975). This diurnal rhythm is independent of the long-term regulation by bile acid

J. B. Schenkman et al. (eds.), Cytochrome P450© Springer-Verlag Berlin Heidelberg 1993

HO~ - HO£ cholesterol 7a-hydroxycholesterol ,

o£ 7a-hydroxy-4-cholesten-3-one

/ , o£ £ 0..-1 ·OH

7a-hydroxy-5~-cholestan-3-one 7a,12a-dihydroxy-4-cholesten-3-one

5~-cholestane-3a, 7 a-diol

~OOH HO·O:X; chenodeoxycholic acid

, H~

O~ 7a,12a-dihydroxy-5~-cholestan-3-one

5~-cholestane-3a,7a,12a-triol , ~OOH

HO·O:X; cholic acid

Fig. 1. Modification of the steroid nucleus by cholesterol 7u-hydroxylase and 12u­hydroxylase

Cholesterol 7a-Hydroxylase and 12a-Hydroxylase 603

feedback (MITROPOULOS et al. 1973). Finally, cytochrome P450ch7u is responsive to short-term modulation by various hormones, drugs and cholesterol precursors, e.g., mevalonate (GIELEN et al. 1976; MITROPOULOS et al. 1978), which can modulate cytochrome P450ch7u activity in a matter of hours.

II. Purification and Properties

BJORKHEM et al. (1974) first solubilized this enzyme from liver microsomes. However, subsequent attempts for purification of the enzyme to homo­geneity by many authors failed with results reflecting the instability and intractability toward purification of this protein. In 1987 the enzyme was eventually purified to homogeneity by Okuda's group (OGISHIMA et al. 1987) using their newly explored assay method (OGISHIMA and OKUDA 1986) and its properties were disclosed. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis (Mf 52000) and revealed the absolute- and dithionite-reduced CO complex absorption spectra character­istic of cytochrome P450. When incubated with the liver microsomal electron transfer system, NADPH and NADPH-cytochrome P450 reductase, it revealed a high activity toward cholesterol (TN 50 min -1) and cholestanol (TN 35 min- 1) but not toward 3~-hydroxy-5-cholenoic acid, taurodeoxycholic acid, cholesteryl acetate, 4-cholesten-3-one, 5~-cholestan-3u-ol and testos­terone. These results suggest that the enzyme does not necessarily require the double bond at C5 but requires a flat structure as realized by the trans- or quasi-trans-fused AlB ring and also requires an equatorial or quasi-equatorial hydroxyl group at C3 . The purified enzyme revealed no activity toward xenobiotics such as 7-ethoxycoumarin and benzo[a]pyrene. The enzyme activity was inhibited neither by amino glutethimide nor by metyrapone, which are well-known inhibitors of cytochromes P45011~ and P450see , respectively. However, the activity was significantly inhibited by iodoacetamide and disulfiram, indicating that the enzyme requires intact sulfhydryl group(s) as suggested by previous authors (BoYD et al. 1973; KALLES and WIKVALL 1981; WAXMAN 1986). Human cholesterol 7-u­hydroxylase was purified by NGUYEN et al. (1990).

Polyclonal antibodies against this enzyme were prepared in mice which were used for isolation of a cDNA clone encoding the enzyme (NOSHIRO et al. 1989). Monospecific polyclonal antibodies were also prepared by other groups (CHIANG et al. 1990; SUNDSETH and WAXMAN 1990).

III. Cloning of cDNA and Gene Structure

A full-length cDNA clone encoding rat liver cholesterol 7u-hydroxylase was first isolated by Okuda's group (NOSHIRO et al. 1989; NOSHIRO et al. 1990). The cDNA contained a 1509-base pair open reading frame encoding 503 amino acid residues corresponding to Mf 56880 and an unusually long

604 K. OKUDA et al.

3'-untranslated region rich in AT sequence in the total length of 3545 base pairs. The predicted amino acid sequence displayed less than 30% similarity to other sequenced cytochrome P450 enzymes. Cytochrome P450ch7a was therefore categorized into a new cytochrome P450 family called CYP7 and its gene into CYP7 (NEBERT et al. 1991). The AT-rich region often con­tained ATITA motifs, 5'-AAT-3' or 5'-TAA-3' trinucleotides, which were reported to be involved in rapidly degrading mRNA such as mRNAs of lymphokines, cytokines and protooncogenes. Complete cDNA clone for human liver cholesterol 7a-hydroxylase was subsequently isolated by NOSHIRO and OKUDA (1990), the deduced amino acid sequence of which showed 82% similarity to rat cytochrome P450ch7a.

The gene encoding this enzyme was isolated from rat genomic DNA by NISHIMOTO et al. (1991). The gene spanned about 11 kilo bases and contained 6 exons. Blotting analysis of genomic DNA and complete matching of restriction maps of several isolated genomic clones indicated that there appeared to be only one gene in the rat genome as suggested by LI et al. (1990). The putative transcription initiation site was present 61 base pairs upstream from the ATG codon. The typical TAT A sequence and CCAAT promoter element were found at 24 and 47 base pairs upstream from the transcription initiation site, respectively. Alignment analysis of several cyto­chrome P450 proteins showed that the cholesterol 7a-hydroxylase gene shared the location of introns with none of the other cytochrome P450 genes except for intron 5, which was in the same position as intron 10 of the gene encoding cytochrome P4504A1 (NEBERT et al. 1991). The alignment analysis also indicated that the distal helix of cholesterol 7a-hydroxylase contained an asparagine in place of the well-conserved threonine that is postulated to be involved in the 02-binding site. Unusual residues, Asn-126 and Thr-442, were also found at the sites where all other cytochromes P450 have positively charged amino acids, which are considered to be involved in interaction with heme propionate. These replacements may be related to the unique function and unusual lability of the hydroxylase. Analysis of evolutionary distance between the cholesterol 7a-hydroxylase gene and other known cytochrome P450 genes indicated that yeast cytochrome P45051a is most closely related to cytochrome P4507a. This finding suggests that the cholesterol 7a­hydroxylase gene (CYP7) is an evolutionarily old cytochrome P450 gene. The structure of the cytochrome P450ch7a gene was also reported by JELINEK and RUSSELL (1990).

IV. Regulation

NOSHIRO et al. (1990) studied a diurnal variation of the levels of the three factors, i.e., enzyme protein, mRNA, and enzyme activity on rat livers prepared at various times of the day, employing the specific antibodies and the cDNA as probes. In normal animals, all three factors exhibited a maximum level at 10:00 p.m. No significant sexual difference was observed.

Cholesterol7u-Hydroxylase and 12u-Hydroxylase 605

Cholestyramine feeding increased all three factors at 10:00 a.m. close to the maximum levels of the normal rats, but did not show a significant increase at 10:00 p.m. On the contrary, starvation markedly decreased all three factors either at 10:00 a.m. or at 10:00 p.m., while maintaining the diurnal variation. A good correlation of the levels of mRNA to the enzyme activities and the protein levels suggests that pretranslational regulation is most likely a mechanism for long-term and mid-term regulation of cholesterol 7a-hydroxylase. The marked diurnal fluctuation of the amount of protein and the level of mRNA also indicates their rapid turnover in accordance with the previous finding (BROWN and BOYD 1974; DANIELSSON and WIKVALL 1981). The short half-life of mRNA could be correlated with the structure of the 3'-untranslated region of the mRNA characteristic of rapidly degrading mRNA, i.e., abundance of motif, AUUUA, and existence of 5'-AAU-3' or 5'-UAA-3' trinucleotides in single-stranded regions of the secondary structure. Similar but slightly different results were also obtained by other groups (JELINEK et al. 1990; LI et al. 1990; SUNDSETH and WAXMAN 1990). In addition, LI et al. fo'md that feeding of rat with a diet supple­mented with 2% cholesterol for 4 days induced cholesterol 7a-hydroxylase activity twofold, and its protein and mRNA levels were also increased. The treatment of rats with dexamethazone and pregnenolone-16a-carbonitrile drastically reduced cholesterol 7a-hydroxylase activity, protein, and mRNA levels, whereas 3-methylcholanthrene, which is known to induce several cytochromes P450 and increases liver microsomal cytochrome P450 content twofold, had little effect on the mRNA level. JELINEK et al. (1990) have shown that the presence of chenodeoxycholic acid or cholic acid in the diet suppressed mRNA levels for the enzyme relative to animals maintained on a normal chow diet. Conversely, cholestyramine or cholesterol in the diet induced the cholesterol 7a-hydroxylase mRNA levels. They also observed that mRNA for HMG-CoA synthase demonstrates a similar pattern of suppression in the presence of dietary bile acid and of induction by cholestyramine, whereas cholesterol suppresses hepatic levels of HMG­CoA synthase mRNA. They have also shown that only the liver expressed cholesterol 7a-hydroxylase mRNA. BJORKHEM et al. (1991) have, however, cast doubt on upregulation due to cholesterol based on their observation that rats treated with cholesterol, less than 1 % in the diet, revealed no significant increase in the enzyme activity compared to those treated with cholesterol, 2% in the diet. These authors hypothesized that a high dietary load of cholesterol caused increased binding of bile acids in the intestine and increased loss of bile acids in feces, which leads to a reduced suppression of the enzyme by the bile acids. PANDAK et al. (1991) observed that chronic biliary diverted rats exhibited a 4.2-fold increase in cholesterol 7a­hydroxylase activity, a 4.5-fold increase in enzyme mass, a lO-fold increase in steady-state mRNA levels and a 3.4-fold increase in transcriptional (nuclear "run-on") activity as compared to nonoperated controls. Intra­duodenal infusion of taurocholate at a rate of 36 ~mol/100 g per hour for

606 K. OKUDA et al.

48 h in these rats caused a significant decrease of these values. They thus suggested that bile acids, either directly or indirectly, downregulate in vivo transcription of the cholesterol 7a-hydroxylase gene, which is probably the major mechanism regulating the levels of this enzyme. HYLEMON et al. (1991) observed that in a serum-free chemically defined medium there was a rapid decrease in the steady-state levels of cholesterol 7a-hydroxylase mRNA in primary culture cells isolated from adult rat liver. The addition of both dexamethasone and T3 (but not the individual addition of each) increased steady-state mRNA levels about 14-fold, suggesting that gluco­corticoid and thyroid hormones play an important role in regulating cholesterol 7a-hydroxylase in vivo.

These results indicate that the regulation of cholesterol 7a-hydroxylase by long-term, mid-term and short-term mechanisms is controlled primarily through alteration of the expression of the underlying protein and mRNA of cytochrome P450ch7a and that some of the mechanisms proposed, i.e., allosteric inhibition, changes in phosphorylation state and changes in the concentration of free cholesterol, are likely to play less significant roles. However, no report has so far been published on the identification of bile acid-sensitive element, which binds to a bile acid-binding trans-acting factor and may result in the repression of the gene expression.

c. 12a-Hydroxylase

I. Physiological Significance

This enzyme catalyzes 12a-hydroxylation of 7a-hydroxy-4-cholesten-3-one to give 7a,12a-dihydroxy-4-cholesten-3-one, which is a precursor of the primary bile acid, cholic acid (EINARSSON 1968). However, 7a-hydroxy-4-cholesten-3-one is also converted to chenodeoxycholic acid, another kind of primary bile acid, if not hydroxylated at the 12a-position (BJORKHEM 1985). The ratio of chenodeoxycholic acid: cholic acid is kept constant in normal rat at 1: 4 but varies in some abnormal conditions such as diabetes (NERVI et al. 1978).

II. Assay Method

The enzyme activity had been assayed using radioactive 7a-hydroxy-4-cholesten-3-one (EINARSSON 1968). The chemical synthesis of this substrate was elaborate and of low yield (BJORKHEM et al. 1965). SHIMASUE (1974) exploited a completely different method to synthesize this compound partly employing enzymatic reaction. Subsequently, NOSHIRO et al. (1985) intro­duced an extremely convenient assay method which uses nonradioactive compound as substrate.

ALLI and ELLIOT (1971) have shown that 5a-cholestane-3a,7a-diol is a better substrate for the 12a-hydroxylase than 7a-hydroxy-4-cholesten-

Cholesterol 7u-Hydroxylase and 12u-Hydroxylase 607

3-one. The fact that allochenodeoxycholic acid is hydroxylated, while chenodeoxycholic acid is not, seems to suggest that the enzyme requires a flat structure as is required in the case of cholesterol 7a-hydroxylase.

III. Purification and Properties

The enzyme was purified to apparent homogeneity by MURAKAMI et al. (1982). However, no report has so far been published about the structure of the enzyme. ISHIDA et al. (1991) recently purified the enzyme to a greater extent using anion exchange high-performance liquid chromatography and prepared polyclonal antibody against this enzyme. The purified enzyme required cytochrome bs in addition to NADPH-cytochrome P4S0 reductase for full activity. NH2-Terminal amino acid sequence is different from those of any P4S0s so far reported and has no methionine residue at NH2-

terminus (ISHIDA et al., 1992). Curiously, however, the activity of the purified enzyme was relatively insensitive to carbon monoxide (BERNHARDSSON et al. 1973; OKUDA et al. 1980).

Based on the known structural requirement of the 12a-hydroxylase, SHAW and ELLIOT (1979) prepared competitive inhibitors with different sub­stitions in the C12 position. The best inhibitor was found to be Sa-cholest-ll­en-3a,7a,26-triol. Theoretically, such inhibitors may be usable to increase the endogenous formation of chenodeoxycholic acid in connection with dissolution of gallstones.

IV. Regulation

JOHANSSON (1970) have found that the enzyme activity is enhanced about fivefold by starving rats. However, it was recently shown that the enzyme is distinct from cytochrome P4S02E1, a well-known alcohol-inducible form of cytochrome P4S0 which is also inducible by starvation (HANSSON 1989). The activity of 12a-hydroxylase is increased by thyroidectomy (MITROPOULOS et al. 1968; BJORKHEM et al. 1973), whereas it is decreased when thyroid hormone is administered. The enzyme activity may also be inhibited by feeding with bile acids (DANIELSSON 1973; AHLBERG et al. 1980). Previously it was shown that the ratio of chenodeoxycholic acid: cholic acid is markedly decreased in the bile of diabetic rats that were treated with streptozotocin (NERVI et al. 1978). However, direct measurement of the enzyme activity using 7a-hydroxy-4-cholesten-3-one as substrate showed that it is somewhat decreased in diabetic rats (KIMURA and OGURA 1988). On the other hand, production of radioactive cholic acid from radioactive Sp-cholestane-3a,7a­diol increased severalfold (KIMURA et al. 1988). These results seem to endorse that 12a-hydroxylase toward 7a-hydroxy-4-cholesten-3-one is distinct from that toward Sp-cholestane-3a,7a-diol as suggested by MURAKAMI et al. (1982). DANIELSSON and KALLES (1983) have reported that the activity of 12a-hydroxylation of Sp-cholestane-3a,7a-diol was stimulated by protein

608 K. OKUDA et al.

factor contained in microsomes, whereas it was inhibited by the cytosolic factor.

D. Concluding Remarks Purification of cholesterol 7a-hydroxylase and isolation of cDNA encoding the enzyme paved the way to study the regulation of cholesterol catabolism at the molecular level. Subsequent studies carried out employing Western and Northern blotting techniques have well established that the regulation of cholesterol 7a-hydroxylase is controlled primarily through alteration in the expression of the underlying protein and mRNA of cytochrome P450ch7a. Recent elucidation of the structure for the gene of this enzyme may also pave the way to analyze the 5' upstream region of the genomic DNA responsible for regulation of gene expression. Such a study together with those for other cytochromes P450 as well as other enzymes than cytochrome P450 involved in cholesterol catabolism will bring an insight to understanding the mechanisms of regulation that underlie the conversion of cholesterol to bile acids.

Acknowledgement. The authors wish to thank Prof. Ingemar Bjorkhem in Karolinska Institute for his critical reading of the manuscript and helpful discussion.

References

Ahlberg J, Angelin B, Bjorkhem I, Einarsson K, Gustafsson J-A, Rafter J (1980) Effects of treatment with chenodeoxycholic acid on liver microsomal metabolism. J Lab Clin Med 95:188-194

Ali SS, Elliot WH (1971) Bile acids. LI. Formation of 12a-hydroxyl derivatives and companions from Sa-sterols by rabbit liver microsomes. J Lipid Res 17:386-392

Bemhardsson C, Bjorkhem I, Danielsson H (1973) 12a-Hydroxylation of 7a­hydroxy-4-cholesten-3-one by a reconstituted system from rat. Biochem Biophys Res Commun 54:1030-1038

Bjorkhem I (1985) In. Danielsson H, Sjovall J, eds "Sterols and Bile Acids" Elsevier, Amsterdam, p 231

Bjorkhem I, Danielsson H, Issidorides C, Kallner A (1965) On the synthesis and metabolism of cholest-4-en-7a-ol-3-one. Acta Chern Scand 19:2151-2154

Bjorkhem I, Danielsson H, Gustafsson J-A (1973) 7a-Hydroxylation of cholesterol by reconstituted systems from rat liver microsomes. FEBS Lett 31:20-22

Bjorkhem I, Danielsson H, Wikvall K (1974) 7a-Hydroxylation of cholesterol by reconstituted systems from rat liver microsomes. Biochem Biophys Res Commun 61:934-941

Bjorkhem I, Eggertsen G, Andersson U (1991) On the mechanism of stimulation of cholesterol 7a-hydroxylase by dietary cholesterol. Biochim Biophys Acta 1085:329-335

Boyd GS, Grimwade AM, Lawson ME (1973) Studies on rat liver microsomal cholesterol 7a-hydroxylase. Eur J Biochem 37:334-340

Brown MJG, Boyd GS (1974) The specificity of the rat-liver cholesterol 7a­hydroxylase. Eur J Biochem 44:37-47

Chiang JYL, Miller WF, Lin G-M (1990) Regulation of cholesterol 7a-hydroxylase in the liver. J BioI Chern 265:3889-3897

Cholesterol 7a-Hydroxylase and 12a-Hydroxylase 609

Danielsson H (1973) Influence of dietary bile acids on formation of bile acids in rat. Steroids 22:667-676

Danielsson H , Wikvall K (1981) Evidence for a specific cytochrome P-450 with short half-life catalyzing 7a-hydroxylation of cholesterol. Biochem Biophys Res Commun 103:46-51

Danielsson H, Kalles I, Lidstr6m B, Lundell K, Wikvall K (1983) Regulation of hydroxylations in biosynthesis of biule acids. Biochem Biophys Res Commun 113:212-219

Einarsson K (1968) On the properties of the 12a-hydroxylase in cholic acid biosynthesis. Eur J Biochem 5:101-108

Gielen JE, Van Cantfort JV, Robaye B, Renson J (1975) Rat-liver cholesterol 7a-hydroxylase 3. New results about its circadian rhythm. Eur J Biochem 55:41-48

Gielen JE, Cantfort J, Kremers P (1976) Genetic and hormonal regulation of steroid hydroxylases and drug metabolizing enzymes in rat liver. Arch Toxicol 36:255-266

Hansson R (1989) Effect of diabetes, starvation, ethanol and isoniazid on rat liver microsomal 12a-hydroxylase activity involved in bile acid biosynthesis. Biochem PharmacoI38:3386-3389

Hylemon PB, Heuman DM, Pandak WM, Chiang JYL, Vlahcevic ZR (1991) Proceedings of the 9th Nobel conference on biosynthesis, regulation and products of the mevalonate pathway; S6dergarn, Sweden, 2-5 June 1991 (in press)

Ishida H, Noshiro M, Okuda K, Coon MJ (1992) Purification and characterization of 7a-hyddroxy-4-cholesten-3-one 12a-hydroxylase. J Bioi Chern 267:21319-21323

Jelinek DF, Russell DR (1990) Structure of the rat gene encoding cholesterol 7a-hydroxylase. Biochemistry 29:7781-7785

Jelinek DF, Andersson S, Slaughter CA, Russel DW (1990) Cloning and regulation of cholesterol 7a-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis. J BioI Chern 265:8190-8197

Johansson G (1970) Effect of cholestyramine and diet on hydroxylation in the biosynthesis and metabolism of bile acids. Eur J Biochem 17:292-295

Kalles I, Wikvall (1981) Role of sulfhydryl groups in catalytic activity of purified cholesterol 7a-hydroxylase system from rabbit and rat liver microsomes. Biochem Biophys Res Commun 100:1361-1369

Kimura K, Ogura Y (1988) Increased rate of cholic acid formation from 3a,7a­dihydroxy-5~-cholestane in perfused livers from diabetic rats. Biochim Biophys Acta 963:329-332

Li YC, Wang DP, Chiang JYL (1990) Regulation of cholesterol 7a-hydroxylase. J Bioi Chern 265:12012-12019

Mitropoulos KA, Suzuki M, Myant NB, Danielsson H (1968) Effect of thyroidectomy and thyroxine on the activity of 12a-hydroxuylase and some components of microsomal electron transfer chains in rat liver. FEBS Lett 1:13-15

Mitropoulos KA, Balasubramaniam S, Myant B (1973) The effect of interuption of the enterophecatic circulation of bile acids and of cholesterol feeding on cholesterol 7a-hydroxylase in relation to the diurnal rhythm in its activity. Biochim Biophys Acta 326:428-438

Mitropoulos KA, Balasubramaniam S, Venkatesan S, Reeves BEA (1978) On the mechanism for the regulation of 3-hydroxyl-3-methylglutaryl-coenzyme A reductase, of cholesterol 7a-hydroxylase and acyl-coenzyme A: cholesterol acyltransferase by the free cholesterol. Biochim Biophys Acta 530:99-111

Murakami K, Okada Y, Okuda K (1982) Purification and characterization of 7a­hydroxuy-4-cholesten-3-one 12a-hydroxylase. J Bioi Chern 257:8030-8035

Myant NB, Mitropoulos KA (1977) Cholesterol 7a-hydroxylase. J Lipid Res 18:135-153

Nebert DW, Nelson DR, Coon MJ, Estabrook RW, Feyereisen R, Fujii-Kuriyama Y, Gonzalez FJ, Guengerich FP, Gunsalus IC, Hohnson EF, Loper JC, Sato R,

610 K. OKUDA et al.: Cholesterol 7u-Hydroxylase and 12u-Hydroxylase

Waterman MW, Waxman DJ (1991) The P450 superfamily. DNA and Cell BioI 10:1-14

Nervi FO, Severin CH, and Valdivieso VD (1978) Bile acid pool changes and regulation of cholate synthesis in experimental diabetes. Biochim Biophys Acta 529:212-223

Nguyen LB, Shefer S, Sal en G, Ness G, Tanaka RD, Packin V, Thomas P, Shore V, Batta A (1990) Purification of cholesterol 7u-hydroxylase from human and rat liver and production of inhibiting polyconal antibodies. J BioI Chern 265:4541-4546

Nishimoto M, Gotoh 0, Okuda K, Noshiro M (1991) Structural analysis of the gene encoding rat cholesterol 7u-hydroxylase, the key enzyme for bile acid biosynthesis. J BioI Chern 266:6467-6471

Noshiro M, Okuda K (1990) Molecular cloning and sequence analysis of cDNA encoding human cholesterol 7u-hydroxylase. FEBS Lett 268:137-140

Noshiro M, Ishida H, Hayashi S, Okuda K (1985) Assays for cholesterol 7u-hydroxylase and 12u-hydroxylase using high performance liquid chromatography. Steroids 45:539-550

Noshiro M, Nishimoto M, Morohashi K, Okuda K (1989) Molecular cloning of cDNA for cholesterol 7u-hydroxylase from rat liver microsomes. FEBS Lett 257:97-100

Noshiro M, Nishimoto M, Okuda K (1990) Rat liver cholesterol 7u-hydroxylase. J BioI Chern 265:10036-10041

Ogishima T, Okuda K (1986) An improved method for assay of cholesterol 7u­hydroxylase activity. Anal Biochem 158:228-232

Ogishima T, Deguchi S, Okuda K (1987) Purification and characterization of cholesterol 7u-hydroxylase from rat liver microsomes. J BioI Chern 262:7646-7650

Okuda K, Atsuta Y, Murakami K, Wakeshima C (1980) In: Coon MJ, Conney AH, Estabrook RW, Gelboin HV, Gillette JR, O'Brien PJ (eds) Microsomes, drug oxidations, and chemical carcinogenesis, vol 2, Academic, New York, p 745-748

Pandak WM, Li YC, Chiang JYL, Studer EJ, Gurley EC, Heuman DM, Vlahcevic ZR, Hylemon PB (1991) Regulation of cholesterol 7u-hydroxylase mRNA and transcriptional activity by taurocholate and cholesterol in the chronic biliary diverted rat. J BioI Chern 266:3416-3421

Reinher E, Bjorkhem I, Angelin B, Ewerth S, Einarsson K (1989) Bile acid synthesis in humans: regulation of hepatic microsomal cholesterol 7u­hydroxylase. Gastroenterology 97: 1498-1505

Shaw R, Elliot WH (1979) Competitive inhibitors of rabbit hepatic microsomal 12u-steroid hydroxylase. J BioI Chern 254:7177-7182

Shimasue A (1974) The action of cholesterol:oxygen oxidoreductase on cholest-5-ene-313,7a-diol and the enzymatic preparation of the labeled cholest-4-en-7u-ol. Hiroshima J Med Sci 23:265-272

Shefer S, Hauser S, Berkersky I, Mosbach EH (1969) Feedback regulation of bile acid biosynthesis in the rat. J Lipid Res 10:646-655

Sundseth SS, Waxman DJ (1990) Hepatic P-450 cholesterol 7u-hydroxylase. J BioI Chern 265: 15090-15095

Waxman DJ (1986) Rat hepatic cholesterol 7u-hydroxylase. Arch Biochem Biophys 247:335-345