Properties of liver microsomal cholesterol 5,6-oxide hydrolase

Cholestane 3 beta,5 alpha, 6 beta-triol has been identified as the exclusive product formed on hydration of cholesterol 5,6 alpha- and 5,6 beta-oxide catalyzed by cholesterol oxide hydrolase in liver microsomes obtained from five mammalian species. Highest activities were present in microsomes from rats and humans. Both acid- and base-catalyzed hydrolysis of the two epoxides also produce this product, presumably due to preference for pseudo-axial opening of the oxirane ring to form product with a trans-AB ring junction. Although the beta-oxide is more reactive than the alpha-oxide upon acid-catalyzed hydration, the alpha-oxide is a 4.5-fold better substrate than the beta-oxide as indicated by values of Vmax/Km. The kinetic parameters Vmax and Km for the reaction catalyzed by rat liver microsomes are 1.68 +/- 0.15 X 10(-7) M min-1 and 10.6 +/- 1.5 microM for the alpha-oxide and 1.32 +/- 0.11 X 10(-7) M min-1 and 37.2 +/- 5.5 microM for the beta-oxide at 0.35 mg protein/ml, pH 7.4, 6.35% (v/v) CH3CN, and 37 degrees C. Several imino compounds are competitive inhibitors for the enzyme from rat liver. The most effective of these is 5,6 alpha-iminocholestanol (Ki = 0.085 microM) which was known to be a good inhibitor from previous studies. Inhibition by aziridines is consistent with the participation of acid catalysis in the mechanism of action of the enzyme. Cholesterol oxide hydrolase is a distinct enzyme from oxidosqualene cyclase as well as microsomal epoxide hydrolase (EC 3.3.2.3) and the recently reported mouse hepatic microsomal epoxide hydrolase that catalyzes the hydration of trans-stilbene oxide.     

Isolation and various properties of alpha-aminocaprolactam hydrolase from Klebsiella aerogenes]

L-alpha-aminocaprolactam hydrolase possessing the L lysine amidase activity was isolated from Klebsiella aerogenes and purified. The procedure of enzymes purification included cell destruction on USDN-I, fractionation by ammonium sulfate, gel chromatography on G-200. The preparation of the purified enzyme possessed specific activity of 50 mumol of lysin per 1 mg of protein per hour. Km was 2.6 mM in case of phosphate buffer (ph 7.2) for I-alpha-aminocaprolactam. Besides L-alpha-aminocaprolactam the enzyme hydrolyses lysine amide, leucine amide tryptophanamide. Magnesium ions are necessary for manifestation of catalytic activity of the enzyme.     

Bile acid synthesis in the Smith-Lemli-Opitz syndrome: effects of dehydrocholesterols on cholesterol 7alpha-hydroxylase and 27-hydroxylase activities in rat liver.

The Smith-Lemli-Opitz syndrome (SLOS) is a congenital birth defect syndrome caused by a deficiency of 3beta-hydroxysterol Delta(7)-reductase, the final enzyme in the cholesterol biosynthetic pathway. The patients have reduced plasma and tissue cholesterol concentrations with the accumulation of 7-dehydrocholesterol and 8-dehydrocholesterol. Bile acid synthesis is reduced and unnatural cholenoic and cholestenoic acids have been identified in some SLOS patients. To explore the mechanism of the abnormal bile acid production, the activities of key enzymes in classic and alternative bile acid biosynthetic pathways (microsomal cholesterol 7alpha-hydroxylase and mitochondrial sterol 27-hydroxylase) were measured in liver biopsy specimens from two mildly affected SLOS patients. The effects of 7- and 8-dehydrocholesterols on these two enzyme activities were studied by using liver from SLOS model rats that were treated with the Delta(7)-reductase inhibitor (BM15.766) for 4 months and were comparable with more severe SLOS phenotype in plasma and hepatic sterol compositions. In the SLOS patients, cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase were not defective. In BM15.766-treated rats, both enzyme activities were lower than those in control rats and they were competitively inhibited by 7- and 8-dehydrocholesterols. Rat microsomal cholesterol 7alpha-hydroxylase did not transform 7-dehydrocholesterol or 8-dehydrocholesterol into 7alpha-hydroxylated sterols. In contrast, rat mitochondrial sterol 27-hydroxylase catalyzed 27-hydroxylation of 7- and 8-dehydrocholesterols, which were partially converted to 3beta-hydroxycholestadienoic acids. Addition of microsomes to the mitochondrial 27-hydroxylase assay mixture reduced 27-hydroxydehydrocholesterol concentrations, which suggested that 27-hydroxydehydrocholesterols were further metabolized by microsomal enzymes. These results suggest that reduced normal bile acid production is characteristic of severe SLOS phenotype and is caused not only by depletion of hepatic cholesterol but also by competitive inhibition of cholesterol 7alpha-hydroxylase and sterol 27-hydroxylase activities by accumulated 7- and 8-dehydrocholesterols. Unnatural bile acids are synthesized mainly by the alternative pathway via mitochondrial sterol 27-hydroxylase in SLOS.     

Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate

Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.     

Catalytic properties and inhibition of hepatic cholesterol-epoxide hydrolase

Hepatic cholesterol-epoxide hydrolase is a microsomal enzyme which appears to be catalytically distinct from the epoxide hydrolase responsible for the catabolism of a wide variety of aromatic and aliphatic epoxides. The diastereomeric forms of cholesterol epoxide, cholesterol 5 alpha,6 alpha-, and cholesterol 5 beta,6 beta-epoxides are converted to cholestane-3 beta,5 alpha,6 beta-triol with equal facility. Kinetic analysis of cholesterol-epoxide hydrolase demonstrated that both diastereomers bind to a common catalytic site. Apparent Km values of 3.69 and 4.42 microM were derived for cholesterol 5 alpha,6 alpha- and cholesterol 5 beta,6 beta-epoxide, respectively. In addition, enzyme activity with both diastereomers was product-inhibited by cholestanetriol through a competitive mechanism with the apparent Ki for cholestanetriol being 10.8 and 6.8 microM against cholesterol alpha- and beta-epoxides, respectively. This inhibitory effect of cholestanetriol may account for the difference observed in the hydration rates for the cholesterol epoxide isomers when they are incubated together in the presence of liver microsomes. Inhibitors of epoxide hydrolase were studied, and three oxidation products were found to be particularly effective against cholesterol-epoxide hydrolase while producing no significant inhibition of styrene-epoxide hydrolase. These inhibitors were 7-ketocholesterol, 6-ketocholestanol, and 7-ketocholestanol, the latter displaying an apparent Ki lower than the Km for either cholesterol epoxide isomer. None of the xenobiotic epoxide hydrolase inhibitors or activators studied affected cholesterol-epoxide hydrolase activity.     

Characterization of acyl-CoA:cholesterol acyltransferase from neonatal chick liver

Endogenous cholesterol esterification in chick liver microsomes was catalyzed by acyl-CoA:cholesterol acyltransferase using palmitoyl-CoA as substrate. An acyl-CoA hydrolase activity was also found in our microsomal preparations. Acyltransferase activity was stable after microsomes storage at -40 degrees C for 6 weeks and increased linearly with the preincubation time between 0 and 45 min. In our assay conditions, cholesteryl ester formation was linear up to 0.3 mg of microsomal protein in the reaction vial and 10 min of incubation. Maximal activity was found in reactions carried out in the presence of 1-2 mM dithiothreitol and 1.2 mg of bovine serum albumin, while acyl-CoA hydrolase was clearly inhibited by increasing albumin amounts.     

In vitro activation of a soluble cholesterol esterase from bovine adrenals by a cAMP-dependent protein kinase.

Properties and partial purification of the bovine adrenal cholesterol esterase from the 100000 X g supernatant fraction were investigated. Variations of the enzyme activity with time-dependent (enzymatic) and time-dependent (non enzymatic) effects have been demonstrated. Mg2 has been proved to inhibit the enzyme activity by a non-enzymatic effect in 50mM Tris/HCl buffer, pH 7.4. A time-dependent inactivation of the cholesterol esterase has been observed in the same buffer. The enzyme could be protected from this enzymatic inactivation by its substrate, cholesterol oleate. cAMP, ATP and Mg2 cuase a time-dependent stimulation of the enzyme in 50mM Tris/HCl buffer, pH 7.4. This result suggests that corticotropin activates the soluble cholesterol esterase from bovine adrenals via cAMP-dependent protein kinase. This view is strengthened by the incorporation of 32P radioactivity from [gamma-32P] ATP into the protein fraction of the 100,000 X g supernatant. The protein-bound 32P radioactivity could be co-purified with the enzyme activity during the partial purification of the soluble cholesterol esterase.     

Purification and characterization of delta 4-3-ketosteroid 5 beta-reductase

delta 4-3-Ketosteroid 5 beta-reductase was purified about 230-fold from 100,000 X g supernatant of rat liver homogenate using 7 alpha-hydroxy-4-cholesten-3-one as substrate throughout. The purified enzyme was electrophoretically homogeneous, and its molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 37,000 and that determined by gel filtration chromatography on calibrated Sephadex G-100 column was 37,200. The absorption spectrum of the purified enzyme showed only a peak at 276 nm due to aromatic amino acids, precluding the presence of a prosthetic group such as flavine in the molecule. The enzyme is highly labile in a low buffer concentration, but is markedly stabilized in the presence of 20% glycerol in 10 mM phosphate buffer. Higher buffer concentration such as 300 mM potassium phosphate buffer was also effective to prevent deterioration in the absence of glycerol, but the effect was somewhat lower compared to glycerol. The purified enzyme showed the activity toward a variety of substrates including testosterone, cortisol, cortisone, progesterone, 4-androstene-3,17-dione, 7 alpha-hydroxy-4-cholesten-3-one, and 7 alpha,12 alpha-dihydroxy-4-cholesten-3-one. The optimal pH for the 5 beta-reduction of 7 alpha-hydroxy-4-cholesten-3-one was 7.4, and the cofactor required for the reaction was NADPH, while NADH revealed no effect. The enzyme activity was inhibited by p-chloromercuribenzoate, but its inhibition was prevented by the presence of a reduced form of glutathione.     

Influence of buffer composition, membrane lipids and proteases on the kinetics of reconstituted cytochrome-c oxidase from bovine liver and heart

Isolated cytochrome-c oxidases from bovine heart and liver were reconstituted in liposomes with asolectin and the kinetics of cytochrome c oxidation were measured under various uncoupled conditions. With 40 mM KCl, 10 mM Hepes, pH 7.4, the liver enzyme showed a higher Vmax in the polarographic but a lower Vmax in the photometric assay. With 125 mM phosphate buffer at pH 6.0 both enzymes revealed identical kinetics. Reconstitution with pure phosphatidylcholine leads to a low activity, which is specifically stimulated for the heart enzyme by inclusion of 10% cardiolipin. Proteoliposomes of both enzymes prepared with asolectin have a high activity, which is unaffected by cardiolipin. Exchanging the intraliposomal buffer, Hepes, for phosphate causes an opposite change of the Vmax and a similar change of the Km for both enzymes suggesting a conformational change of the extraliposomal binding domain for cytochrome c through the membrane. Proteases change the kinetics of both enzymes, but to a different degree. The data indicate a complex and tissue-specific influence of nucleus-coded subunits on the catalytic activity of cytochrome-c-oxidase.     

The biological conversion of 7-dehydrocholesterol into cholesterol and comments on the reduction of double bonds

It is shown that the 7-dehydrocholesterol reductase-catalysed conversion of 7-dehydrocholesterol into cholesterol (II), with a 105000g microsomal pellet of rat liver in the presence of [4-(3)H(2)]NADPH, results in the transfer of radioactivity to the 7alpha-position of cholesterol. When the conversion is carried out in the presence of tritiated water the label is introduced exclusively at the 8beta-position. However, when the conversion of 7-dehydrocholesterol into cholesterol is performed with a 500g supernatant of rat liver homogenate the radioactivity is incorporated at both the 7alpha- and the 8beta-position. Evidence is provided for the presence of an enzyme system in the 500g supernatant that catalyses an equilibration of hydrogen atoms between those at the 4-position of NADPH and those of water. The work with stereospecifically labelled cofactors shows that both the equilibrating system and the 7-dehydrocholesterol reductase utilize the 4B-hydrogen atom of NADPH. In the light of these results a mechanism for the reduction of carbon-carbon double bonds is discussed.     

Leukotriene A4 hydrolase: a zinc metalloenzyme

Purified human leukotriene A4 hydrolase is shown to contain 1 mol of zinc per mol of enzyme, as determined by atomic absorption spectrometry. The enzyme is inhibited dose-dependently by the chelating agents 8-hydroxy-quinoline-5-sulfonic acid, and 1,10-phenanthroline with KI values of about 2 and 8 x 10(-4) M, respectively, whereas dipicolinic acid and EDTA are ineffective in this respect. The inhibition by 1,10-phenanthroline is time-dependent, and at a concentration of 5 mM, 50% inhibition of enzyme (3 x 10(-7) M) occurs after about 15 min. The zinc atom of leukotriene A4 hydrolase can be removed by dialysis against 1,10-phenanthroline which results in loss of enzyme activity. The catalytic activity is almost completely restored by the addition of stoichiometric amounts of Zn2+ or Co2+.     

Mechanistic studies on carboxypeptidase A from goat pancreas: role of arginine residue at the active site

Chemical modification of carboxypeptidase Ag1 from goat pancreas with phenylglyoxal or ninhydrin led to a loss of enzymatic activity. The inactivation by phenylglyoxal in 200 mM N-ethylmorpholine, 200 mM sodium chloride buffer, pH 8.0, or in 300 mM borate buffer, pH 8.0, followed pseudo-first-order kinetics at all concentrations of the modifier. The reaction order with respect to phenylglyoxal was 1.68 and 0.81 in 200 mM N-ethylmorpholine, 200 mM NaCl buffer and 300 mM borate buffer, pH 8.0, respectively, indicating modification of single arginine residue per mole of enzyme. The kinetic data were supported by amino acid analysis of modified enzyme, which also showed the modification of single arginine residue per mole of the enzyme. The modified enzyme had an absorption maximum at 250 nm, and quantification of the increase in absorbance showed modification of single arginine residue. Modification of arginine residue was protected by beta-phenylpropionic acid, thus suggesting involvement of an arginine residue at or near the active site of the enzyme.     

Purification and characterization of 7 alpha-hydroxy-4-cholesten-3-one 12 alpha-monooxygenase

7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase was purified from liver microsomes of phenobarbital-treated rabbits. The purification was carried out by solubilization of microsomes by cholate, fractionation with polyethylene glycol, affinity chromatography on cholate-Sepharose 4B column, hydroxylapatite column chromatography, chromatography on DEAE-Sepharose CL-6B column, and a second hydroxylapatite column chromatography. The purified preparation gave a single major band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and contained 9.0 nmol of cytochrome P-450/mg of protein, which corresponded to 5.3-fold purification from microsomes on the basis of specific heme content. The specific activity of the enzyme expressed as enzyme activity per mg of enzyme protein was increased 315-fold from microsomes. The molecular weight of the enzyme was estimated to be 56,000 from calibrated polyacrylamide gel electrophoresis. The enzyme-pH curve gave a peak at pH 7.0. The Michaelis constant for 7 alpha-hydroxy-4-cholesten-3-one was 27 microM. Absorption spectra of the oxidized form of the enzyme showed a Soret band at 418 nm. 7 alpha-Hydroxy-4-cholesten-3-one 12 alpha-monooxygenase activity was reconstituted from the purified cytochrome P-450, NADPH-cytochrome P-450 reductase, dilauroylglyceryl-3-phosphorylcholine, and NADPH. The purified enzyme was free from steroid 25-hydroxylase activity and that of 26- or 27-hydroxylase but revealed some activity for benzphetamine N-demethylation. The enzyme activity was not inhibited by metapyrone, aminoglutethimide, and KCN, but was seriously inhibited by nonionic detergents such as Emulgen 913. The enzyme was labile under low buffer concentrations but was stabilized at least for 4 weeks under higher buffer concentration such as 300 mM phosphate buffer.     

Loss of cytochrome P-450 from hepatic nuclear membranes of rats fed 2-acetylaminofluorene

The cytochrome P-450 content of nuclear membranes isolated from the livers of male Sprague-Dawley rats fed a semipurified diet containing 0.05% w/w 2-acetylaminofluorene (AAF) for 3 weeks, was only about 20% of the values in control rats fed the same diet devoid of AAF. This effect was apparent after only 1 week of AAF treatment and persisted in nuclear membranes from isolated hyperplastic nodules (HPN) generated by 4 cycles of interrupted AAF-feeding. The microsomal cytochrome P-450 content, on the other hand, remained at control levels after 1 week of AAF treatment, and it was only slightly decreased after 3 weeks. In contrast, microsomes from HPN generated by prolonged AAF treatment had markedly decreased amounts of cytochrome P-450. The AAF treatment also caused changes in cholesterol epoxide hydrolase activity, which paralleled those observed for cytochrome P-450 content. Nuclear membranes from livers of rats fed AAF for 3 weeks, and from isolated HPN, had only 30-50% of the cholesterol epoxide hydrolase activity present in controls, whereas the microsomal enzyme activity remained at control levels after 3 weeks of AAF feeding but was 50% depressed in microsomes from HPN. The selective loss of cytochrome P-450 and of cholesterol epoxide hydrolase in hepatic nuclear membrane, but not in microsomes, of rats fed AAF for 3 weeks suggests independent control for these enzymes in these two membrane fractions. Cytochrome P-450 plays a role both in the activation of AAF (N-hydroxylation) as well as in its detoxification (ring hydroxylation) whereas cholesterol epoxide hydrolase initiates the detoxification of cholesterol epoxide. Therefore, our findings suggest the hypothesis that AAF treatment causes an early loss, at the surface of the nucleus, of the last line of defense for detoxification of transforming or promoting metabolites generated by microsomal activation of natural substances such as cholesterol and of xenobiotics such as AAF.     

Mitochondrial adenosine triphosphatase from human placenta--purification and catalytic properties

1. The purification of ATPase (EC 3.6.1.3) from human placental mitochondria is described. The yield based on mitochondrial enzyme activity was about 70% and the purification was 380-fold. 2. The rate of Mg-ATP hydrolysis was 85 mumole per min per mg of protein under optimum conditions. 3. Nucleoside triphosphates were hydrolyzed by the purified enzyme at decreasing rates in the following order: GTP greater than ITP greater than ATP greater than epsilon-ATP greater than UTP greater than CTP in Tris-HCl buffer (pH 8.0), and in the order: ATP greater than GTP greater than or equal to ITP greater than epsilon-ATP greater than UTP greater than CTP in Tris-bicarbonate buffer at pH 8.0. 4. The values of kinetic parameters are reported. The ATPase reaction deviated from typical Michaelis-Menten kinetics in Tris-HCl buffer but not in Tris-bicarbonate. Eadie-Hofstee plots for Mg-ATP hydrolysis were biphasic in Tris-HCl (Km = 0.2 mM, 0.09 mM) and monophastic in Tris-becarbonate medium (Km = 0.16 mM). 5. In the presence of Mg-ITP or Mg-GTP as substrates no curvature of the reciprocal plots was observed. 6. The results presented reflect the fact that multiple conformations of the enzyme molecule do exist and are probably involved in its regulatory functions. 7. The existence of two kinetically distinct classes of catalytic sites and of an anion-binding site on the placental ATPase is proposed.     

Enzymatic Mechanisms Involved in Phenanthrene Degradation by the White Rot Fungus Pleurotus ostreatus

The enzymatic mechanisms involved in the degradation of phenanthrene by the white rot fungus Pleurotus ostreatus were examined. Phase I metabolism (cytochrome P-450 monooxygenase and epoxide hydrolase) and phase II conjugation (glutathione S-transferase, aryl sulfotransferase, UDP-glucuronosyltransferase, and UDP-glucosyltransferase) enzyme activities were determined for mycelial extracts of P. ostreatus. Cytochrome P-450 was detected in both cytosolic and microsomal fractions at 0.16 and 0.38 nmol min(sup-1) mg of protein(sup1), respectively. Both fractions oxidized [9,10-(sup14)C]phenanthrene to phenanthrene trans-9,10-dihydrodiol. The cytochrome P-450 inhibitors 1-aminobenzotriazole (0.1 mM), SKF-525A (proadifen, 0.1 mM), and carbon monoxide inhibited the cytosolic and microsomal P-450s differently. Cytosolic and microsomal epoxide hydrolase activities, with phenanthrene 9,10-oxide as the substrate, were similar, with specific activities of 0.50 and 0.41 nmol min(sup-1) mg of protein(sup-1), respectively. The epoxide hydrolase inhibitor cyclohexene oxide (5 mM) significantly inhibited the formation of phenanthrene trans-9,10-dihydrodiol in both fractions. The phase II enzyme 1-chloro-2,4-dinitrobenzene glutathione S-transferase was detected in the cytosolic fraction (4.16 nmol min(sup-1) mg of protein(sup-1)), whereas aryl adenosine-3(prm1)-phosphate-5(prm1)-phosphosulfate sulfotransferase (aryl PAPS sulfotransferase) UDP-glucuronosyltransferase, and UDP-glucosyltransferase had microsomal activities of 2.14, 4.25, and 4.21 nmol min(sup-1) mg of protein(sup-1), respectively, with low activity in the cytosolic fraction. However, when P. ostreatus culture broth incubated with phenanthrene was screened for phase II metabolites, no sulfate, glutathione, glucoside, or glucuronide conjugates of phenanthrene metabolites were detected. These experiments indicate the involvement of cytochrome P-450 monooxygenase and epoxide hydrolase in the initial phase I oxidation of phenanthrene to form phenanthrene trans-9,10-dihydrodiol. Laccase and manganese-independent peroxidase were not involved in the initial oxidation of phenanthrene. Although P. ostreatus had phase II xenobiotic metabolizing enzymes, conjugation reactions were not important for the elimination of hydroxylated phenanthrene.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.     

Effects of Tween 80 on liver microsomal 7-dehydrocholesterol reductase

Enzymatic conversion of 7-dehydrocholesterol to cholesterol by liver microsomes was increased by addition of Tween 80. This increase was proportional to Tween 80 concentration, and reached its maximum of 250% of baseline activity after addition of 300 μg/ml of Tween 80. This enhancement was comparable to that achieved by addition of 7.4 mg/ml of cytosol protein. No additive effect was observed with Tween 80 combined with cytosol protein. These data suggest that Tween 80 can substitute for sterol carrier protein 2 in the conversion of 7-dehydrocholesterol to cholesterol.     

The effects of metyrapone, chalcone epoxide, benzil, clotrimazole and related compounds on the activity of microsomal epoxide hydrolase in situ, in purified form and in reconstituted systems towards different substrates

The influence of metyrapone, chalcone epoxide, benzil and clotrimazole on the activity of microsomal epoxide hydrolase towards styrene oxide, benzo[a]pyrene 4,5-oxide, estroxide and androstene oxide was investigated. The studies were performed using liver microsomes from rats, rabbits, mice and humans; epoxide hydrolase purified from rat liver microsomes to apparent homogeneity; and the purified enzyme incorporated into liposomes composed of egg-yolk phosphatidylcholine or total rat liver microsomal lipids. All four effectors were found to activate the hydrolysis of styrene oxide by epoxide hydrolase in situ in rat liver microsomal membranes, in agreement with earlier findings. Epoxide hydrolase activity towards styrene oxide in liver microsomes from mouse, rabbit and man was also increased by all four effectors. The most striking effect was a 680% activation by clotrimazole in rat liver microsomes. However, none of the effectors activated microsomal epoxide hydrolase more than 50% when benzo[a]pyrene 4,5-oxide, estroxide or androstene oxide was used as substrate. Indeed, clotrimazole was found to inhibit microsomal epoxide hydrolase activity towards estroxide 30-50% and towards androstene oxide 60-90%. The effects of these four compounds were found to be virtually identical in the preparations from rats, rabbits, mice and humans. The effects of metyrapone, chalcone epoxide, benzil and clotrimazole on purified epoxide hydrolase were qualitatively the same as those on epoxide hydrolase in intact microsomes, but much smaller in magnitude. These effects were increased in magnitude only slightly by incorporation of the purified enzyme into liposomes made from egg-yolk phosphatidylcholine. However, when incorporation into liposomes composed of total microsomal lipids was performed, the effects seen were essentially of the same magnitude as with intact microsomes. When the extent of activation was plotted against effector concentration, three different patterns were found with different effectors. Activation of epoxide hydrolase activity towards styrene oxide by clotrimazole was found to be uncompetitive with the substrate and highly structure specific. On the other hand, inhibition of epoxide hydrolase activity towards androstene oxide by clotrimazole was found to be competitive in microsomes. It is concluded that the marked effects of these four modulators on microsomal epoxide hydrolase activity are due to an interaction with the enzyme protein itself, but that the presence of total microsomal phospholipids allows the maximal expression leading to similar degrees of modulation as those observed in intact microsomes.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subcellular distribution, catalytic properties and partial purification of epoxide hydrolase in the human adrenal gland

Epoxide hydrolase in human adrenal gland was characterized with respect to catalytic properties and subcellular distribution. With human adrenal microsomes and the substrates styrene-7,8-oxide, cis-stilbene oxide, estroxide and androstene oxide the specific activities were between 1.9 and 19.0 nmol/min/mg protein. With styrene-7,8-oxide as substrate the apparent Km-value was 0.98 mM and the pH optimum was 9.2. Subcellular fractionation revealed that the bulk of the activity was confined to the endoplasmic reticulum. Different compounds known to influence rodent microsomal epoxide hydrolase activity were also tested on the human adrenal enzyme. 1,1,1-Trichloropropene-2,3-oxide (TCPO) and cyclohexene oxide (CHO) inhibited the activity while benzil and clotrimazole stimulated the activity. Partial purification of human adrenal epoxide hydrolase indicates that its molecular weight is about 51 000 and that its concentration relative total protein in the human adrenal microsomes is about 10%.