Biosynthesis of hydroxyl-linked glucuronides of short-chain bile acids by rat liver 3-hydroxysteroid UDP-glucuronosyltransferase

Microsomal preparations from livers of Sprague-Dawley rats catalyze the glucuronidation of 3 alpha-hydroxy-5 beta-H (3 alpha, 5 beta) short-chain bile acids (C20-C23), predominantly at the hydroxyl group, while the glucuronidation of 3 beta, 5 beta short-chain bile acids occurs exclusively at the carboxyl group. A similar pattern of conjugation was also observed in Wistar rats having normal levels of 3-hydroxysteroid UDP-glucuronosyltransferase. Significant reductions of formation rates for hydroxyl-linked, but not carboxyl-linked, short-chain bile acid glucuronides were observed in hepatic microsomes from Wistar rats with low 3-hydroxysteroid UDP-glucuronosyltransferase activity. 3-Hydroxysteroid UDP-glucuronosyltransferase, purified to homogeneity from Sprague-Dawley liver microsomes, catalyzed the 3-O-glucuronidation of 3 alpha, 5 beta C20-23 bile acids, as well as of lithocholic and isolithocholic acids (C24). The apparent Michaelis constants (KM) for short-chain bile acids were similar to the value obtained for androsterone. 3 alpha, 5 beta-C20 and 3 beta, 5 beta-C20 competitively inhibited glucuronidation of androsterone by the purified 3-hydroxysteroid UDP-glucuronosyltransferase. Purified 17 beta-hydroxysteroid and p-nitrophenol UDP-glucuronosyltransferases did not catalyze the glucuronidation of bile acids. In addition, none of the purified transferases catalyzed the formation of carboxyl-linked bile acid glucuronides. The results show that 3-hydroxysteroid UDP-glucuronosyltransferase, an enzyme specific for 3-hydroxyl groups of androgenic steroids and some conventional bile acids, also catalyzes the glucuronidation of 3 alpha-hydroxyl (but not carboxyl) groups of 3 alpha, 5 beta short-chain bile acids.     

Isolation and characterization of hyodeoxycholic-acid: UDP-glucuronosyltransferase from human liver

The enzyme hyodeoxycholic-acid: UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine--Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of Mr 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6 alpha-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13:1:2.7. UDP-glucuronosyltransferase activities toward the 3 alpha-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine--Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent Km values of 12 microM and 4 microM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.     

Novel inhibitors and substrates of bilirubin: UDP-glucuronosyltransferase. Arylalkylcarboxylic acids

The in vitro inhibitory potency of 20 structurally related alkanoic and arylalkanoic acids has been investigated on rat liver UDP-glucuronosyltransferase. These compounds were tested on the microsomal and purified enzyme, and a cloned cDNA expressed in COS 7 cell cultures. Among all the acids tested, 7,7,7-triphenylheptanoic acid was the most powerful inhibitor of bilirubin:UDP-glucuronosyltransferase with a lower effect on 1-naphtol, androsterone and testosterone glucuronidation. The inhibition was competitive towards the microsomal and purified bilirubin:UDP-glucuronosyltransferases with Kiapp values of 12.0 microM and 1.6 microM, respectively. Twenty analogues were examined, and the results showed that their inhibitory potency on bilirubin:UDP-glucuronosyltransferase activity was a function of at least three structural features (a) the presence of a hydrophobic triphenyl moiety; (b) the length of the aliphatic chain and (c) the presence of a carboxylic group. These inhibitors were also tested as possible substrates of UDP-glucuronosyltransferases. The strongest inhibitors were poor substrates of rat liver microsomal UDP-glucuronosyltransferases. However, 7,7,7-triphenylheptanoic acid was actively glucuronidated by purified bilirubin:UDP-glucuronosyltransferase, in contrast to its analogues with decreasing alkyl chain length. In addition, glucuronidation of this molecule was enhanced by clofibrate treatment but could not be detected in Gunn rats, which are deficient in bilirubin:UDP-glucuronosyltransferase, further indicating that the glucuronidation of this compound was catalysed by bilirubin:UDP-glucuronosyltransferase. The results suggest that 7,7,7-triphenylheptanoic acid may be a useful structural probe to investigate the molecular basis of glucuronidation of bilirubin and carboxylic acids.     

Effects of steroid hormones and xenobiotics on the pubertal development of UDP-glucuronosyltransferase activities towards androsterone and 4-nitrophenol in Wistar rats.

Oestradiol benzoate, testosterone propionate, progesterone, corticosterone, 3-methylcholanthrene and phenobarbital were administered to Wistar rats at the pubertal period, and their effects on hepatic UDP-glucuronosyltransferase activities were determined. Pretreatment with oestradiol benzoate had a temporary suppressive effect on androsterone UDP-glucuronosyltransferase activity in rats with the high-activity phenotype of androsterone glucuronidation. The effect was marked in 40-day-old rats, but was not found in older rats. Androsterone UDP-glucuronosyltransferase activity was induced by phenobarbital in rats with the high-activity phenotype, but not in rats with the low-activity phenotype. Foster-feeding experiments showed that breast milk did not alter the genetically determined expression of androsterone UDP-glucuronosyltransferase activity in Wistar rats. In contrast, 4-nitrophenol UDP-glucuronosyltransferase activity was not affected by steroid hormones, but was highly induced by 3-methylcholanthrene.     

The enzymatic mechanism of glucuronidation catalyzed by two purified rat liver steroid UDP-glucuronosyltransferases

A kinetic analysis of two homogeneous rat liver steroid (3 alpha-hydroxysteroid and 17 beta-hydroxysteroid) UDP-glucuronosyltransferases was conducted using bisubstrate kinetic analysis, product inhibition studies, and dead-end competitive inhibition studies. Double reciprocal plots of initial velocity versus substrate concentration, using bisubstrate kinetic analysis, gave a sequential mechanism. Product inhibition studies were compatible with either a rapid equilibrium, random-order kinetic mechanism or an ordered Theorell-Chance mechanism. Results of dead-end competitive inhibition studies excluded an ordered Theorell-Chance mechanism. The cumulative results are consistent with a rapid equilibrium random-order sequential kinetic mechanism for the glucuronidation of testosterone by purified 17 beta-hydroxysteroid UDP-glucuronosyltransferase and of androsterone by purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.     

Strain differences in rat liver (UDP-glucuronyltransferase activity towards androsterone.

Male Donryu, Wistar King rats showed discontinuous variations in hepatic microsomal UDP-glucuronyltransferase activities towards androsterone, but not towards testosterone, bilirubin, phenolphthalein and 4-nitrophenol. Fresh microsomal fraction with a low transferase activity towards androsterone formed 0.049--0.080 nmole of glucuronide/min per mg of protein, whereas fresh microsomal fraction with a high transferase activity towards androsterone formed 0.335--0.557 nmol of glucuronide/min per mg of protein. The microsomal fraction with low enzyme activity towards androsterone was not stimulated by treatment with Triton X-100 or freezing and thawing. In contrast, male Long Evans and Sprague-Dawley rats did not exhibit such diversity.     

Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens.

We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity. The final enzyme preparation was purified 252-fold, with a recovery of 14%. Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000. The isoelectric point was approximately pH 6.1. The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups. The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3. The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate. Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM. The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9. The apparent Km for NAD+ was 526 microM. The initial reaction velocities with NADPH were less than 50% of those with NADH. The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg. These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.     

Aldrin epoxidation catalyzed by purified rat-liver cytochromes P-450 and P-448. High selectivity for cytochrome P-450

Aldrin epoxidation was studied in monooxygenase systems reconstituted from purified rat liver microsomal cytochrome P-450 or P-448, NADPH-cytochrome c reductase, dilauroylphosphatidylcholine and sodium cholate. Cytochrome P-450, purified from hepatic microsomes of phenobarbital-treated rats, exhibited a high rate of dieldrin formation. The low enzyme activity observed in the absence of the lipid and sodium cholate was increased threefold by addition of dilauroylphosphatidylcholine and was further stimulated twofold by addition of sodium cholate. The apparent Km for aldrin in the complete system was 7 +/- 2 microM. SKF 525-A, at a concentration of 250 microM, inhibited aldrin epoxidation by 65%, whereas 7,8-benzoflavone had no inhibitory effect at concentrations up to 250 microM. Addition of ethanol markedly increased epoxidase activity. The increase was threefold in the presence of 5% ethanol. When cytochrome P-448 purified from hepatic microsomes of 3-methylcholanthrene-treated rats was used, a very low rate of epoxidation was observed which was less than 3% of the activity mediated by cytochrome P-450 under similar assay conditions. Enzyme activity was independent of the lipid factor dilauroylphosphatidylcholine. The apparent Km for aldrin was 27 +/- 7 microM. The modifiers of monooxygenase reactions, 7,8-benzoflavone, SKF 525-A and ethanol, inhibited the activity mediated by cytochrome P-448. The I50 was 0.05, 0.2 and 800 mM, respectively. These results indicate that aldrin is a highly selective substrate for cytochrome P-450 species present in microsomes of phenobarbital-treated animals and is a poor substrate for cytochrome P-448. The two forms of aldrin epoxidase can be characterised by their turnover number, their apparent Km and their sensitivity to modifiers, like 7,8-benzoflavone and ethanol.     

Developmental alteration of hepatic UDP-glucuronosyltransferase and sulphotransferase towards androsterone and 4-nitrophenol in Wistar rats.

Postnatal development of hepatic UDP-glucuronosyltransferase and sulphotransferase activities towards androsterone and 4-nitrophenol as well as cytochrome P-450 contents was studied in male and female Wistar rats. The rats with high and low UDP-glucuronosyltransferase activity towards androsterone were classified by the genotype of the parent animals. UDP-glucuronosyltransferase activity towards androsterone began rapidly to enhance after 30 days of age in the high-activity group, whereas the transferase activity remained low throughout in the low-activity group. Such a striking difference was not observed in UDP-glucuronosyltransferase activity towards 4-nitrophenol, sulphotransferase activity towards androsterone and 4-nitrophenol, and cytochrome P-450 contents. Sex-based difference in the sulphotransferase activity was marked after 30 days of age. Sulphotransferase activity towards androsterone was much higher in adult females than in adult males, whereas higher sulphation activity towards 4-nitrophenol was found in adult males. The results also indicate that the low level of the UDP-glucuronosyltransferase activity did not lead to compensatory stimulation of the sulphotransferase activity.     

Purification and characterization of an ethanol-induced UDP-glucuronosyltransferase

A UDP-glucuronosyltransferase (GT) enzyme was isolated from ethanol-induced male New Zealand white rabbit hepatic protein. The animals were pretreated for 2 weeks with 10% ethanol in their drinking water. The GT enzyme was purified by anion-exchange and affinity chromatography and was shown to be homogeneous by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The molecular mass of the ethanol-induced UDP-glucuronosyltransferase was determined to be 57,000 Da. Tryptic digests of the ethanol-induced GT and a similarly purified GT from control rabbit liver appeared to be different by HPLC analysis, even though the molecular masses of the enzymes were indistinguishable. Amino acid compositions of the two proteins were different for six amino acids. The apparent Km values for the ethanol-induced GT enzyme for 1-naphthol and morphine as substrates were 43 and 109 microM, respectively. The apparent Vmax values for the ethanol-induced GT enzyme for these substrates were 83 and 4.6 nmol/min/mg protein. The increases in catalytic efficiencies, apparent Vmax/Km for 1-naphthol and for morphine, for the ethanol-induced isozyme compared to the control isozyme activities were 2.0- and 2.4-fold. A polyclonal antibody raised in sheep to the rabbit ethanol-induced GT demonstrated a 520-fold selectivity for precipitation of the ethanol-induced protein rather than the control protein. These results demonstrate the production of an unique isozyme of UDP-glucuronosyltransferase that is produced in rabbits as a result of chronic ethanol exposure.     

Hepatic and renal conjugation (Phase II) enzyme activities in young adult, middle-aged, and senescent male Sprague-Dawley rats

Acetaminophen (APAP)-induced nephrotoxicity is age dependent in male Sprague-Dawley rats: nephrotoxicity occurs at lower dosages of APAP in 12- to 14-month olds compared with 2- to 3-month olds. The mechanisms responsible for enhanced nephrotoxicity in 12-month-old Sprague-Dawley rats are not entirely clear, but may be related to age-dependent differences in APAP metabolism in liver and/or kidney. Major pathways of hepatic APAP metabolism include sulfation and glucuronidation; glutathione conjugation represents a pathway for detoxification of reactive oxidative APAP metabolites. The present studies were designed to quantify in vitro activity of three Phase II enzyme activities: glutathione S-transferase using 1-chloro-2,4-dinitrobenzene as substrate, UDP-glucuronyl transferase using APAP as substrate, and sulfotransferase using APAP as substrate, in subcellular fractions of liver and kidney of 3-, 12-, 18-, and 30-month-old naive male Sprague-Dawley rats. In liver, glutathione S-transferase, UDP glucuronyl transferase, and sulfotransferase activities were not significantly different in rats from 3 through 30 months of age. Renal UDP glucuronyl transferase and sulfotransferase activities were similar in rats from 3 through 30 months of age. In contrast, renal glutathione S-transferase activity was characterized by a lower Km in 12- and 30-month olds when compared with 3-month olds. These data suggest that the reduced total systemic clearance of APAP in 12-month-old male Sprague-Dawley rats previously observed cannot be attributed to age-dependent differences in hepatic APAP metabolism. In addition, it is unlikely that differences in renal APAP metabolism contribute to age-dependent APAP nephrotoxicity.     

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.     

Purification of the NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase from female rat pituitary cytosol

The NADPH:5 alpha-dihydroprogesterone 3 alpha-hydroxysteroid oxidoreductase (3 alpha-HSOR) [EC 1.1.1.50] which catalyzes the reversible conversion of 5 alpha-pregnane-3,20-dione (5 alpha-dihydroprogesterone; 5 alpha-DHP) to 3 alpha-hydroxy-5 alpha-pregnan- 20-one (3 alpha-,5 alpha-tetrahydroprogesterone; 3 alpha,5 alpha-THP) was purified to apparent homogeneity from female rat anterior pituitary cytosol by a three step micro-purification procedure. Specific activity of purified 3 alpha-HSOR was enriched 438-fold from that in pituitary cytosol using successive ion exchange, chromatofocusing and affinity column chromatography purification steps. 3 alpha-HSOR appears to be a monomer with an approximate molecular weight of 36 kDa and an isoelectric point of about 5.75. The purified enzyme appears as a single protein staining band (36 kDa) when examined by polyacrylamide gel electrophoresis and with both silver or Coomassie blue staining. Under non-dissociating electrophoretic conditions, all of the 3 alpha-HSOR activity co-migrated with the 36 kDa protein staining band. The purified enzyme in the presence of the preferred cofactor, NADPH, has an apparent Km for 5 alpha-DHP of 82 nM and a Vmax of 1.2 mumol of 3 alpha,5 alpha-THP formed per mg protein/30 min. The Km for NADPH was 0.71 microM. In the oxidative direction, the enzyme in the presence of NADP+ has a Km for 3 alpha,5 alpha-THP of 1.4 microM and a Vmax of 9.7 mumol of 5 alpha-DHP formed per mg protein/30 min. The Km for NADP+ was 1.6 microM.     

Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5′-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

Large deletion of androsterone UDP-glucuronosyltransferase gene in the inherited deficient strain of Wistar rats.

LA Wistar rats have a deficiency of androsterone UDP-glucuronosyltransferase (UDPGT) and are present in Wistar rat colonies around the world. In order to clarify the molecular mechanism of the deficiency, androsterone UDPGT cDNA clone, pGT2 was isolated from rat liver cDNA library and was digested with restriction enzymes to afford three probes for Northern and Southern blot analyses in HA (normal), heterozygous LA and LA Wistar rats. In Northern blot analysis, androsterone UDPGT mRNA was totally absent in LA Wistar rat liver. Southern blot analysis suggested a large deletion of androsterone UDPGT gene in the rats. Genomic DNA amplifications with synthetic primers which have nucleotide sequences corresponding to the 5'-region of androsterone UDPGT cDNA, suggested that androsterone UDPGT gene has exon 1 with a length of some 700 bp and that this exon is deleted in LA Wistar rats. Based on these lines of evidence, it is concluded that the large portion of androsterone UDPGT gene is deleted in LA Wistar rats, which results in the absence of androsterone UDPGT mRNA and consequently the corresponding enzyme protein.     

The molecular basis of the inherited deficiency of androsterone UDP-glucuronyltransferase in Wistar rats

A major UDP-glucuronyltransferase isoenzyme in rat liver (51 kDa), corresponding to androsterone glucuronidating activity, has been identified by immunoblot analysis. This isoenzyme is absent from Wistar rats exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity exhibiting the low androsterone (LA) UDP-glucuronyltransferase activity phenotype. Northern blot analysis of total RNA from normal and androsterone glucuronidation deficient Wistar rats demonstrated that the mRNA encoding this protein was not synthesised. Differences in restriction fragment length observed on Southern blotting of genomic DNA from LA Wistar rats indicate that this inherited deficiency is the result of a deletion in the androsterone UDP-glucuronyltransferase gene.     

Purification and properties of 3 alpha-hydroxysteroid dehydrogenase from rat brain cytosol. Inhibition by nonsteroidal anti-inflammatory drugs and progestins

The 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) of rat brain cytosol has been purified to apparent homogeneity. The purification procedure involves six successive steps, includes one affinity chromatography, and yields enzyme which displays a 1,550-fold enhancement in specific activity. The homogeneous enzyme has a Km of 8.0 microM for 5 alpha-dihydrotestosterone, a Vmax of 1.3 mumol of 3 alpha-androstanediol formed per h/mg of protein, and displays a preference for NADPH. It appears to be the major activity responsible for the reduction of 5 alpha-dihydrotestosterone in this tissue and may play a pivotal role in brain androgen metabolism. The homogeneous enzyme has several properties in common with the 3 alpha-hydroxysteroid dehydrogenase purified from rat liver cytosol (Penning, T. M., Mukharji, I., Barrows, S., and Talalay, P. (1984) Biochem. J. 222, 601-611). It is a monomer with a molecular weight of 31,000, it has a pI of 5.5, and it is potently inhibited by the nonsteroidal anti-inflammatory drugs (IC50 value for indomethacin = 2.0 microM). The potency of inhibition observed for the brain enzyme parallels that observed for cyclooxygenase: indomethacin greater than fenamates greater than l-methylpyrrole acetic acids greater than arylpropionic acids greater than salicylates greater than acetaminophen. Examination of a variety of steroidal contraceptives as modulators of the dehydrogenase indicates that ethinylestradiol is a very poor inhibitor (IC50 = 100 microM), while 6-medroxyprogesterone acetate (Provera) is an extremely potent inhibitor (IC50 = 0.2 microM). The possibility exists that brain androgen metabolism may be altered by the nonsteroidal anti-inflammatory drugs and synthetic progestins.     

Camphoroquinone reduction: another reaction catalyzed by rat liver cytosol 3 alpha-hydroxysteroid dehydrogenase

A new purification scheme is described for the female rat liver cytosolic enzyme dually catalyzing the oxidation of androsterone (3 alpha-hydroxysteroid:NAD(P)+ oxidoreductase, EC 1.1.1.50) and acenaphthenol (trans-1,2-dihydrobenzene-1,2-diol:NADP+ oxidoreductase, EC 1.3.1.20). This purification procedure yielded the most highly purified preparation of this enzyme thus far published as adjudged from its androsterone oxidation activity. In addition, we have demonstrated that this purified enzyme also catalyzes the reduction of camphoroquinone, a natural monoterpene, non-aromatic quinone. The nature of the products of the camphoroquinone reduction has been partially elucidated and agrees with previously published results (Robertson, J.S. and Solomon, E. (1971) Biochem. J. 121, 503-509). Kinetic studies of the metabolism of androsterone, camphoroquinone and acenaphthenol by the enzyme have been performed, yielding respective Km and Vmax values. The results of these studies allow a clarification of the mechanism of action of this enzyme, particularly with respect to its dihydrodiol dehydrogenase activity.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

Product inhibition of o-aminophenol glucuronidation by p-nitrophenyl glucuronide in detergent-treated microsomal fractions.

The glucuronidation of o-aminophenol is unaffected by p-nitrophenyl gluronide when native microsomal fractions are the source of UDP-glucuronyltransferase. When microsomal fractions treated with Lubrol detergent are the source of the enzyme, however, p-nitrophenyl glucuronide exhibits competitive inhibition of o-aminophenol glucuronidation. In addition, the apparent K1 for p-nitrophenyl glucuronide is the same whether o-aminophenol or p-nitrophenol is the acceptor substrate. The data suggest that UDP-glucuronyltransferase has one binding site for the two phenols and that the absence of inhibition observed in native microsomal fractions is dependent on an intact microsomal membrane.