24-hydroxycholesterol is a substrate for hepatic cholesterol 7alpha-hydroxylase (CYP7A).

(24S)-Hydroxycholesterol is formed from cholesterol in the brain and is important for cholesterol homeostasis in this organ. Elimination of (24S)-hydroxycholesterol has been suggested to occur in the liver but little is known about the metabolism of this oxysterol. In the present investigation, we report formation of 7alpha, 24-dihydroxycholesterol in pig and human liver. 7alpha-hydroxylase activity toward both isomers of 24-hydroxycholesterol [(24S) and (24R)] was found in a partially purified and reconstituted cholesterol 7alpha-hydroxylase (CYP7A) enzyme fraction from pig liver microsomes. In contrast, a purified enzyme fraction of pig liver oxysterol 7alpha-hydroxylase with high activity toward 27-hydroxycholesterol did not show any detectable activity toward 24-hydroxycholesterol. 7alpha-Hydroxylation of 24-hydroxycholesterol was strongly inhibited by 7-oxocholesterol, a known inhibitor of CYP7A. Human CYP7A, recombinantly expressed in Escherichia coli and in simian COS cells, showed 7alpha-hydroxylase activity toward both cholesterol and the two isomers of 24-hydroxycholesterol, with a preference for the (24S)-isomer. Our results show that 24-hydroxycholesterol is metabolized by CYP7A, an enzyme previously considered to be specific for cholesterol and cholestanol and not active toward oxysterols. Because CYP7A is the rate-limiting enzyme in the major pathway of bile acid biosynthesis, the possibility is discussed that at least part of the 24-hydroxycholesterol is converted into 7alpha-hydroxylated bile acids by the enzymes involved in the normal biosynthesis of bile acids.

Cholesterol 7alpha-hydroxylase activities from human and rat liver are modulated in vitro posttranslationally by phosphorylation/dephosphorylation.

Purified cholesterol 7alpha-hydroxylases (C7alphaH) from human and rat liver microsomes, and from transformed Escherichia coli expression systems, were incubated with 0.3 mmol/L [gamma-32P] adenosine triphosphate (ATP) in the presence and absence of bacterial alkaline phosphatase (AP) or rabbit muscle adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. The amounts of 32P incorporation after separation of human and rat C7alphaH proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were related to C7alphaH catalytic activities (determined by a radioisotope incorporation method) and enzyme protein mass (determined by Western blotting and laser densitometry). Both human and rat C7alphaH activities significantly decreased after dephosphorylation by AP (-57% - -72%) and increased up to twofold with phosphorylation by rabbit muscle cAMP-dependent protein kinase. The increases in C7alphaH activities were proportional to the amounts of cAMP-dependent protein kinase used, and were coupled to 32P incorporation into the purified enzymes. Both the activation of C7alphaH and the amounts of 32P incorporation were time-dependent and reached a maximum after 1 hour of incubation with 5 U of cAMP-dependent protein kinase. In a second set of experiments, purified human and rat liver C7alphaH were dephosphorylated by 30-minute incubation with AP, followed by inactivation of the phosphatase by the inhibitor NaF, and rephosphorylation of C7alphaH by 30-minute incubation with rabbit muscle cAMP-dependent protein kinase or bovine heart cAMP-independent protein kinase. Rephosphorylation of the dephosphorylated C7alphaH proteins by cAMP-dependent protein kinase increased C7alphaH catalytic activities up to fourfold, and the stimulation in catalytic activities paralleled the increases in 32P incorporation into the purified enzymes. Bovine heart protein kinase was as potent as rabbit muscle cAMP-dependent protein kinase in stimulating catalytic activity and 32P incorporation into the human C7alphaH protein. Because the protein mass of these purified enzymes did not change, the short-term regulation or catalytic efficiency of C7alphaH (activity per protein mass unit) is modulated, in vitro, posttranslationally by a phosphorylation/dephosphorylation mechanism in both the human and the rat enzymes.

Expression and purification of human cholesterol 7 alpha-hydroxylase in Escherichia coli.

Cholesterol 7 alpha-hydroxylase (P450c7) is the first and rate-limiting enzyme in bile acid biosynthesis and is the product of a cytochrome P450 gene, CYP7. We have previously reported the cloning of a full-length human cholesterol 7 alpha-hydroxylase cDNA (Karam, W. G., and J. Y. L. Chiang. 1992. Biochem. Biophys. Res. Commun. 185: 588-595). Using this clone in a polymerase chain reaction, we have generated a cDNA (H7 alpha 1.5) in which the codons for the N-terminal 24 amino acid residues were deleted. The translational product of this cDNA would be a truncated protein, P450c7(delta 2-24) with a hydrophilic NH2-terminal sequence, Met-Ala-Arg-Arg-Arg-Gln... This cDNA was cloned into the expression vector pJL and the construct pJL/H7 alpha 1.5 was transformed into E. coli strain TOPP3. We have also ligated a truncated rat cholesterol 7 alpha-hydroxylase cDNA obtained previously (Li, Y. C., and J. Y. L. Chiang. 1991. J. Biol. Chem. 266: 19186-19191) into the pJL vector and have transformed this construct (pJL/R7 alpha 1.5) into E. coli strain MV1304. Both of these systems expressed functional cholesterol 7 alpha-hydroxylase in E. coli. A fivefold improvement in the expression of rat enzyme over the previous expression system was obtained. About 70-80% of the truncated human P450 in the clear lysate was localized in the cytosol. The truncated human and rat P450c7(delta 2-24) were purified to homogeneity. Reconstitution of cholesterol 7 alpha-hydroxylase activity using purified rat or human P450c7(delta 2-24) showed a similar Km of 6 and 7 microM for cholesterol, a Vmax of 0.13 and 0.14 nmol/min, and a turnover number of 1.3 and 1.5 per min, respectively. Immunoblotting experiment revealed that a polyclonal antibody raised against rat microsomal cholesterol 7 alpha-hydroxylase recognized both rat and human P450c7(delta 2-24). This expression system provides a method for isolation of a large quantity of purified and catalytically active cholesterol 7 alpha-hydroxylase for the study of structure and function of this important enzyme in bile acid synthesis and cholesterol homeostasis.

Partial cloning of cDNA for hamster liver cholesterol 7 alpha-hydroxylase.

A partial cDNA clone encoding cholesterol 7 alpha-hydroxylase (pHA 7 alpha-3) was isolated from a hamster liver cDNA library using rat P-450 ch7 cDNA (p 7 alpha-11) as a probe. In comparison with the complete base sequence of p 7 alpha-11, pHA 7 alpha-3 lacked about 500 base pairs at the 5' end. Circadian rhythms of mRNA for hamster liver 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase were investigated with cDNA clones encoding 3-hydroxy-3-methylglutaryl coenzyme A reductase (pRed-227) and pHA 7 alpha-3. Typical circadian rhythms of mRNA for both enzymes and a time lag between the two circadian rhythms were observed. The circadian rhythm of activity for each enzyme corresponded to that of each mRNA.

Cloning and regulation of cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme in bile acid biosynthesis.

The rate-limiting step in bile acid biosynthesis is catalyzed by the microsomal cytochrome P-450 cholesterol 7 alpha-hydroxylase (7 alpha-hydroxylase). The expression of this enzyme is subject to feedback regulation by sterols and is thought to be coordinately regulated with enzymes in the cholesterol supply pathways, including the low density lipoprotein receptor and 3-hydroxy-3-methylglutaryl-coenzyme A reductase and synthase. Here we report the purification of rat 7 alpha-hydroxylase and the determination of a partial amino acid sequence. Oligonucleotides derived from peptide sequence were used to clone a full-length cDNA encoding 7 alpha-hydroxylase. DNA sequence analysis of the cDNA revealed a 7 alpha-hydroxylase protein of 503 amino acids with a predicted molecular weight of 56,890 which represents a novel family of cytochrome P-450 enzymes. Transfection of a 7 alpha-hydroxylase cDNA into simian COS cells resulted in the synthesis of a functional enzyme whose activity was stimulated in vitro by the addition of rat microsomal cytochrome P-450 reductase protein. RNA blot hybridization experiments indicated that the mRNA for 7 alpha-hydroxylase is found only in the liver. The levels of this mRNA increased when bile acids were depleted by dietary cholestyramine and decreased when bile acids were consumed. Dietary cholesterol led to an increase in 7 alpha-hydroxylase mRNA levels. The enzymatic activity of 7 alpha-hydroxylase paralleled the observed changes in mRNA levels. These results suggest that bile acids and sterols are able to alter the transcription of the 7 alpha-hydroxylase gene and that this control explains the previously observed feedback regulation of bile acid synthesis.

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.

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.

Effect of diabetes on partially purified hepatic cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme of bile acid biosynthesis.

The effect of diabetes on the activity of hepatic cholesterol 7 alpha-hydroxylase (CH-7 alpha) was studied in reconstituted systems using partially purified cytochrome P-450 (P-450) from control and diabetic rat livers. With both preparations, the rate of product formation increased when the amount of P-450 in the incubations was increased. However, the increase in the rate of product formation was greater with the control P-450. When varying levels of dithiothreitol (DTT) were added to the incubations, the specific activity of the control P-450 was found to be directly proportional to the concentration of DTT. However, DTT did not have any consistent effect on the specific activity of the diabetic P-450. Finally, while 25 mM NaF in the incubation medium had minimal effect on the specific activity of the control P-450, the specific activity of the diabetic P-450 increased by 32% in the presence of 25 mM NaF. These studies have shown some important differences in the properties of CH-7 alpha in control and diabetic animals and emphasize the need to study the enzyme under more defined conditions.

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.

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

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

Microsomal enzymes of cholesterol biosynthesis. Purification of lanosterol 14 alpha-methyl demethylase cytochrome P-450 from hepatic microsomes.

Employing reconstitution assays and measurement of cytochrome P-450 content, lanosterol 14 alpha-demethylase and cholesterol 7 alpha-hydroxylase have been studied in solubilized preparations of rat hepatic microsomes. Both activities have been resolved from other cytochrome P-450 isozymes and each other by chromatography on DEAE-Sephacel and adsorption on hydroxylapatite. The demethylase has been further purified to homogeneity by cation exchange chromatography on Mono-S resin. The purified cytochrome displays a specific content of 15.8 nmol of heme/mg of protein and a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 51,000. A Soret maximum for the reduced/CO binding complex at 448 nm is observed. Reconstitution of the purified cytochrome with NADPH-cytochrome-c reductase, dilaurylphosphatidylcholine, NADPH, and O2 supports the demethylation process which is inhibited by CO. Reconstitution also affords accumulation of oxygenated, metabolic intermediates with single catalytic turnover of the cytochrome, thus supporting the hypothesis that a single isozyme of cytochrome P-450 is responsible for all three oxidations and the lyase activity involved in the lanosterol C-32 demethylation sequence. Low oxidase activity toward several xenobiotic substrates and selectivity toward endogenous sterol substrates is observed for the purified cytochrome. These results indicate a high degree of substrate specificity for the cytochrome, which would be expected for a constitutive P-450 involved in anabolic biochemical processes.

Evidence against in vitro modulation of rat liver cholesterol 7 alpha-hydroxylase activity by phosphorylation-dephosphorylation: comparison with hydroxymethylglutaryl CoA reductase.

The activity of cholesterol 7 alpha-hydroxylase in rat liver microsomes was investigated under conditions favourable for phosphorylation-dephosphorylation. The enzyme activity was similar in the presence or absence of sodium fluoride during preparation. Preincubation with ATP and magnesium did not affect the enzyme activity. Cholesterol 7 alpha-hydroxylase was inhibited by alkaline phosphatase, but this inhibition was similar also after inactivation of the phosphatase. Under similar conditions, rat hepatic hydroxymethylglutaryl CoA reductase activity was clearly modulated in agreement with phosphorylation-dephosphorylation. The absence of such a modulation of cholesterol 7 alpha-hydroxylase argues against involvement of phosphorylation-dephosphorylation in the regulation of this enzyme.

Properties of cholesterol 7 alpha-hydroxylase in rat liver microsomal acetone powder.

Rat liver microsomes were extracted with acetone, and a microsomal powder preparation was obtained. The cholesterol 7 alpha-hydroxylase activity of acetone powder was linear with time, the amount of protein, and the amount of cholesterol in human or rat serum. Unesterified lipoprotein cholesterol was also an effective substrate, and the Km values increased progressively from high-density lipoprotein (HDL) to low-density lipoprotein (LDL) to very-low-density lipoprotein (VLDL), suggesting that HDL-free-cholesterol was the better substrate.

Influence of species and sex on cholesterol 7 alpha-hydroxylase activity in experimental animals.

Cholesterol 7 alpha-hydroxylase activity was evaluated measuring by selected ion monitoring the amount of 7 alpha-hydroxycholesterol formed from endogenous microsomal cholesterol and from exogenously added [4-14C]cholesterol in incubations of liver microsomes. The enzyme activity was determined in female rats, hamsters and guinea pigs and results were compared with those obtained in male rats. The results suggest that there is no difference between male and females as far as the activity of the enzyme from endogenous cholesterol is concerned at least in rat liver. Moreover, saturation of the enzyme by the endogenous substrate was observed in all female animals in contrast to the male rat for which the enzyme was found to be not saturated, as already reported by others. From molar radioactivity of 7 alpha-hydroxycholesterol obtained during incubations with [4-14C]cholesterol, differences were also evidenced in the equilibration of the substrate with the endogenous pool of cholesterol. In the male rat, equilibration depended upon the concentration of radioactive cholesterol added to the medium and reached completion at 60 microM concentration. In female animals, molar radioactivity of 7 alpha-hydroxycholesterol was always linearly correlated with that of cholesterol in the medium but it was always lower than that one. This indicates that in the tested female animals only part of exogenously added cholesterol is accessible to the substrate pool of the enzyme.

Hepatic microsomal activities of cholesterol 7 alpha-hydroxylase and 3-hydroxy-3-methylglutaryl-CoA reductase in the prairie dog. An animal model for cholesterol gallstone disease.

The prairie dog is a useful experimental animal model for studies of cholesterol gallstone pathogenesis. The unique susceptibility to rapid induction of gallstones solely by feeding of a 1.2% cholesterol diet in this species could result from low levels of hepatic cholesterol 7 alpha-hydroxylase. With optimal assay conditions in hepatic microsomes, a basal specific activity of about 25 pmol/min per g protein was found. Administration of diets containing 1.2% cholesterol or 5% cholestyramine caused hydroxylase levels to increase 60 and 250%, respectively. This response pattern is similar to that observed in other species under the same conditions, indicating that abnormally low basal or inappropriately unresponsive hydroxylase levels are not susceptibility factors unique to this model. With optimal assay conditions for hydroxymethylglutaryl-CoA reductase, a Km of 32.5 microM (S-HMG-CoA) and basal specific activities of between 60 and 175 pmol/min per mg protein were found. Following feeding of either sodium chenodeoxycholate or sodium cholate, in reasonable pharmacologic doses, no suppression of hydroxylase and reductase levels was found. These findings undermine the widely held view that the therapeutic effect of oral chenodeoxycholate in man for cholesterol gallstone dissolution is directly mediated by suppression of the activities of these enzymes.

Cholesterol 7 alpha-hydroxylase of rat liver. Studies on the solubilisation, resolution and reconstitution of the enzyme complex.

1. Cytochrome P-450 was prepared from the liver microsomes of cholestyramine-fed rats by solubilisation with the non-ionic detergent Nonidet P42 followed by chromatography on a DEAE-cellulose column and on hydroxyapatite. NADPH--cytochrome P-450 reductase was prepared by a technique of affinity column chromatography using 2',5'ADP Sepharose. 2. The activity of cholesterol 7 alpha-hydroxylase was measured in a reconstituted system of microsomal mixed-function oxidase containing cytochrome P-450 and NADPH--cytochrome P-450 reductase from rat liver plus cholesterol and NADPH. Endogenous cholesterol was largely depleted from the enzyme preparations by the treatment of the microsomes with cold n-butanol/acetone. 3. The reconstituted system of mixed-function oxidase catalysed a highly effective and specific 7 alpha-hydroxylation of cholesterol. The reconstituted system showed a higher activity of cholesterol 7 alpha-hydroxylase than was observed in native liver microsomes. The reconstituted system had an absolute requirement for cytochrome P-450, NADPH--cytochrome P-450 reductase and NADPH. 4. The apparent Km for cholesterol in the reconstituted system was 15 microM and the V was 1.4 nmol 7 alpha-hydroxycholesterol formed min-1 (nmol cytochrome P-450)-1. 5. The reconstituted system also catalysed the 7 alpha-hydroxylation of taurodeoxycholic acid, the 7 alpha-hydroxylation of 26-norcholesterol and to a limited degree the 12 alpha-hydroxylation of cholest-4-ene-3-one-7 alpha-ol. The ability of this reconstituted system to effect these two 7 alpha-hydroxylation reactions and the 12 alpha-hydroxylation reaction was significantly less than the ability of the system to 7 alpha-hydroxylate cholesterol.

Cholesterol 7 alpha-hydroxylase from human liver: partial purification and reconstruction into defined phospholipid-cholesterol vesicles.

Cholesterol 7 alpha-hydroxylase, the rate-limiting enzyme for bile acid synthesis, was shown to be copurified with human liver microsomal cytochrome P-450. When these cytochrome P-450 species were reconstituted in phospholipid-cholesterol vesicles together with NADPH-cytochrome P-450 reductase, high cholesterol 7 alpha-hydroxylase activity was obtained in the presence of NADPH. The activity represented a twofold enrichment relative to cytochrome P-450 and 43-fold enrichment relative to total microsomal protein. Availability of such a preparation will allow further characterization of the enzyme and will also allow studies of its mechanisms of regulation.