Inhibition of UDP-glucuronosyltransferase activity by possible transition-state analogues in rat-liver microsomes

A series of possible transition state analogues of the glucuronidation reaction catalyzed by UDP-glucuronosyltransferase were tested for their inhibitory effect on glucuronidation of various substrates in a rat liver microsomal fraction. In general 4-nitrophenol glucuronidation was more effectively inhibited than that of 1-naphthol, bilirubin or testosterone. 2-(1-Naphthyl)ethyl-UDP and 2,2,2-(triphenyl)ethyl-UDP were the most effective inhibitors. Their inhibitory effect was competitive towards both UDP-glucuronic acid and 4-nitrophenol. These compounds were much more effective inhibitors than UDP; therefore addition of a lipophilic group enhances the inhibitory potency of UDP. The various UDP derivatives showed differences in their abilities to inhibit the glucuronidation of the four acceptor substrates, supporting the concept that the different forms of UDP-glucuronosyl transferase have different active sites.     

Purification and immunochemical characterization of a low-pI form of UDP glucuronosyltransferase from mouse liver

A liver UDP glucuronosyltransferase (GT) enzyme from either phenobarbital- or 3-methylcholanthrene-treated C57BL/6N mice was isolated by phenyl-Sepharose, DEAE-ion exchange, and UDP hexanolamine chromatographic steps. This enzyme had a broad substrate specificity and was mainly responsible for the microsomal capacity to glucuronidate testosterone, 1-naphthol, and morphine. This UDP glucuronosyltransferase ( GTM1 ) appeared to be at least 95% homogeneous and had a subunit molecular weight of 51,000 using sodium dodecyl sulfate-polyacrylamide gel and two-dimensional gel electrophoreses. Antibodies prepared against the purified protein developed a single immunoprecipitin line by double-diffusion analysis with purified antigen and with solubilized microsomes from both control and drug-induced C57BL/6N and DBA/2N mice. A precipitin line was also observed with microsomal proteins which isoelectrofocused at approximately pH 6.7, but not with those which isoelectrofocused at approximately pH 8.5. GTM1 was, therefore, designated at low-pI form. Immunopurified antibody preferentially inhibited and immunoprecipitated GT activities toward testosterone, 1-naphthol, and morphine. To a lesser extent, activities toward phenolphthalein, 3-hydroxybenzo[a]pyrene, and estrone were inhibited while activities toward 4-nitrophenol and 4-methylumbelliferone were not affected. All activities, however, were immunoadsorbed in the presence of protein A-Sepharose. This observation can be explained by the following results. Immunoprecipitates from labeled microsomes contained primarily a 51,000-Da protein. When the immune complexes were adsorbed with protein A-Sepharose, a 54,000-Da protein as well as the expected 51,000-Da GTM1 was detected. This 54,000-Da protein was associated with the glucuronidation of 3-hydroxybenzo[a]pyrene and 4-nitrophenol, and was designated GTM2 .     

S-Glucuronidation of 7-mercapto-4-methylcoumarin by human UDP glycosyltransferases in genetically engineered fission yeast cells

Human UDP glycosyltransferases (UGTs) play an important role in xenobiotic detoxification. They increase the solubility of their substrates by adding a sugar moiety (such as glucuronic acid) to different functional entities (such as hydroxyl groups). The aim of this study was to investigate how glucuronidation of a standard substrate is affected by a change of the hetero-atom at the conjugation site. For this purpose, we compared the in vitro glucuronidation rates of 4-methylumbelliferone and 7-mercapto-4-methylcoumarin, respectively. Human liver microsomes catalyzed the S-glucuronidation of 7-mercapto-4--methylcoumarin almost as efficient as the O-glucuronidation of 4-methylumbelliferone. When testing isoenzyme specificity by whole cell biotransformation with fission yeast strains that recombinantly express all 19 human members of the UGT1 and UGT2 families, it was found that 13 isoenzymes were able to glucuronidate 7-mercapto-4-methylcoumarin, with five of them being specific for this substrate and the other eight also converting 4-methylumbelliferone under these conditions. The remaining six UGTs did not accept either substrate. Out of the eight isoenzymes that glucuronidated both substrates, four catalyzed both reactions approximately to the same extent, while three displayed higher conversion rates towards 4-methylumbelliferone and one preferred 7-mercapto-4-methylcoumarin. These data suggest that 7-mercapto-4-methylcoumarin is a convenient new standard substrate for monitoring S-glucuronidation.     

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.     

Purification of a form of mouse liver UDP glucuronosyltransferase which glucuronidates androgens

A form of UDP glucuronosyltransferase active in the glucuronidation of the androgens, testosterone, androsterone and dihydrotestosterone has been purified to apparent homogeneity as judged by sodium dodecylsulfate polyacrylamide gel electrophoresis from the livers of phenobarbital-treated C57BL/6N mice. This UDP glucuronosyltransferase is inactive towards estrone as substrate. Data from chromatofocusing and purification experiments suggest that testosterone and androsterone are glucuronidated primarily by this enzyme form and to a lesser extent by an enzyme form which has a slightly higher isoelectric point. However, this major form is only responsible for about half the capacity to glucuronidate dihydrotestosterone.     

Characterization of a nitrophenol reductase from the phototrophic bacterium Rhodobacter capsulatus E1F1.

The phototrophic bacterium Rhodobacter capsulatus E1F1 photoreduced 2,4-dinitrophenol to 2-amino-4-nitrophenol by a nitrophenol reductase activity which was induced in the presence of nitrophenols and was repressed in ammonium-grown cells. The enzyme was located in the cytosol, required NAD(P)H as an electron donor, and used several nitrophenol derivatives as alternative substrates. The nitrophenol reductase was purified to electrophoretic homogeneity by a simple method. The enzyme was composed of two 27-kDa subunits, was inhibited by metal chelators, mercurial compounds, and Cu2+, and contained flavin mononucleotide and possibly nonheme iron as prosthetic groups. Purified enzyme also exhibited NAD(P)H diaphorase activity which used tetrazolium salt as an electron acceptor.     

Selective and potent inhibition of different hepatic UDP-glucuronosyltransferase activities by omega,omega,omega-triphenylalcohols and UDP derivatives.

A homologous series of omega,omega,omega-triphenylalcohols and corresponding omega,omega,omega-triphenylalkyl-UDP derivatives was synthesized and tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes, with 1-naphthol, testosterone and bilirubin as substrates. Introduction of the UDP moiety in the triphenylalcohols increased their inhibition potency markedly toward the isoforms which glucuronidate 1-naphthol and testosterone, but strongly decreased that toward bilirubin. The inhibiting potency of the UDP-derivatives increased as a function of the length of the hydrocarbon chain. The best inhibitor 7,7,7-triphenylheptyl-UDP showed an I50 of 30 and 10 microM for 1-naphthol and testosterone glucuronidation, respectively; even a 1 mM concentration of the compound had little, if any, effect on bilirubin glucuronidation. The inhibition by 7,7,7-triphenylheptyl-UDP was mixed-type toward 1-naphthol, and non competitive toward testosterone (apparent K(i) 30 microM and 1.7 microM, respectively); on the other hand, the inhibition was competitive toward the common substrate UDP-glucuronic acid (apparent K(i) 1.9-1.2 microM). In addition, 7,7,7-triphenylheptyl-UDP (0.25-0.50 mM) almost inhibited glucuronidation of 1-naphthol and testosterone catalyzed by the recombinant rat liver UGT-2B1 and human liver UGT-1A1, whose cDNA has been expressed in V79 cells. In conclusion, the data indicate that 7,7,7-triphenyheptyl-UDP interacted competitively with the UDP binding site of UGT. The results also indicate that it is possible to design transition state analogue inhibitors with specificity for different UGT forms.     

On the mechanism of the modulation in vitro of acyl-CoA:cholesterol acyltransferase by progesterone.

The assay of acyl-CoA:cholesterol acyltransferase (ACAT) in the presence of progesterone resulted in a lower enzyme activity and this inhibition was dependent on the concentration of steroid in the assay mixture. The incubation at 37 degrees C of rat liver microsomal fraction followed by the re-isolation of treated microsomal vesicles and the assay of ACAT resulted in a pre-incubation-time-dependent increase in the activity of the enzyme. This rate of increase was inhibited by the presence of progesterone in the pre-incubation mixture. The incubation of the microsomal fraction in the presence of cholesterol/phosphatidylcholine liposomes, followed by the re-isolation of the treated microsomal vesicles and assay of ACAT, resulted in time-dependent and liposomal cholesterol-concentration-dependent transfer of cholesterol to microsomal vesicles and in an increase in the activity of ACAT. The presence of progesterone during pre-incubation had no effect on the rate of transfer of liposomal cholesterol to the microsomal vesicles. However, progesterone decreased the rate of change in ACAT activity. This effect can be attributed to progesterone associated with treated microsomal vesicles and present during the enzyme assay. Consistent with this, the presence of progesterone has no effect on the size of the non-esterified cholesterol pool that acts as substrate for ACAT. The size of the ACAT substrate pool was modulated in vitro or in vivo and ACAT activity was assayed in the presence of various concentrations of progesterone. The data suggest that the interaction of the steroid with ACAT is at a site other than the catalytic site and that changes in the size of the substrate pool are associated with an increase in ACAT activity, but do not result in changes in the conformation of the enzyme or in co-operative transitions of the enzyme.     

Studies of mammalian glucoside conjugation

The mammalian glucoside-conjugation pathway was studied by using p-nitrophenol as the model substrate and mouse liver microsomal preparations as the source of enzyme. The microsomal preparations supplemented with UDP-glucose glucosylated p-nitrophenol; p-nitrophenyl glucoside was identified by chromatography in six solvent systems. The unsolubilized glucosyltransferase of fresh microsomal preparations did not follow the usual Michaelis-Menten kinetics and was easily inhibited by many steroids. All the steroids tested inhibited glucosylation of p-nitrophenol to a greater degree than glucuronidation of p-nitrophenol when assayed in the same microsomal preparations. The steroids inhibited glucosylation with the following decreasing effectiveness: pregnan-3alpha-ol-20beta-one (3alpha-hydroxypregnan-20-beta-one)>oestradiol-17beta 3-methyl ether>oestradiol-17beta>oestriol>pregnane-3alpha,20beta-diol>oestrone. Pregnan-3alpha-ol-20beta-one, pregnane-3alpha,20beta-diol and oestrone had negligible effect on glucuronidation.     

The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The K(m) of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.     

Immunochemical characterization of human lung epoxide hydrolases

Immunochemical techniques were used to investigate the biochemical properties of human lung epoxide hydrolases. Two epoxide hydrolases with different immunoreactive properties were identified. These two epoxide hydrolases were found in both cytosolic and microsomal cell fractions. Immunotitration of enzyme activity showed that enzymes that catalyze the hydration of benzo(a)pyrene 4,5-oxide react with antiserum to rat microsomal epoxide hydrolase; those that hydrate trans-stilbene oxide do not. Immunotitration and Western blot experiments showed that microsomal and cytosolic benzo(a)pyrene 4,5-oxide hydrolases have significant structural homology. Immunohistochemical staining of human lung benzo(a)pyrene 4,5-oxide hydrolase showed that the enzyme is localized primarily in the bronchial epithelium. No cell type-specific localization was observed. An enzyme-linked immunosorbent assay was developed which allows direct quantitation of benzo(a)pyrene 4,5-oxide hydrolase protein. Levels of enzyme protein detected by this assay correlated well with enzyme levels determined by substrate conversion assays.     

Regulation of rat-liver glycoprotein: N-acetylneuraminic acid transferase activity by pyrimidine nucleotides.

CMP-N-acetylneuraminic acid: glycoprotein sialyltransferase activities were assayed in rat liver microsomal fractions using desialylated fetuin as the substrate acceptors for N-acetylneuraminic acid. It was found that cytidine nucleotides specifically depressed enzyme activities. CMP was shown to act as a competitive inhibitor with an apparent Ki of 0.62 mM. N-Acetylneuraminic acid at 1.15 mM had no effect on enzyme activities. Uridine nucleotides at 1.15 mM, especially UDP, increased enzyme activities. UDP may act as an allosteric activating agent increasing the apparent V. Other nucleotides, sugars and nucleotide-sugars at similar concentrations affected sialyltransferase activities only slightly. A general mechanism is proposed for the regulation of glycosyltransferase activities by free nucleotides.     

Control of continuous polyhydroxybutyrate synthesis using calorimetry and flow cytometry

The substrate-carbon flow can be controlled in continuous bioreactor cultures by the medium composition, for example, by the C/N ratio. The carbon distribution is optimal when a maximum fraction flows into the desired product and the residual is just sufficient to compensate for the dilution of the microbial catalyst. Undershooting of the latter condition is reflected immediately by changes in the Gibbs energy dissipation and cellular states. Two calorimetric measurement principles were applied to optimize the continuous synthesis of polyhydroxybutyrate (PHB) by Variovorax paradoxus DSM4065 during growth with constantly increasing supply rates of fructose or toxic phenol. Firstly, the changed slope of the heat production rate in a complete heat balanced bioreactor (CHB) indicated optimum carbon channeling into PHB. The extent of the alteration depended directly on the toxic properties of the substrate. Secondly, a flow through calorimeter was connected with the bioreactor as a "measurement loop." The optimum substrate carbon distribution was indicated by a sudden change in the heat production rate independent of substrate toxicity. The sudden change was explained mathematically and exploited for the long-term control of phenol conversion into PHB. LASER flow cytometry measurements distinguished between subpopulations with completely different PHB-content. Populations grown on fructose preserved a constant ratio of two subpopulations with double and quadruple sets of DNA. Cells grown on phenol comprised a third subpopulation with a single DNA set. Rising phenol concentrations caused this subpopulation to increase. It may thus be considered as an indicator of chemostress.     

The stimulation by sodium fluoride of plasma-membrane Ca2+ inflow in isolated hepatocytes. Evidence that a GTP-binding regulatory protein is involved in the hormonal stimulation of Ca2+ inflow.

Bilirubin may be transported within intracellular membranes of the hepatocyte and may undergo membrane-membrane transfer to gain access to the conjugating enzyme UDP-glucuronyltransferase in the endoplasmic reticulum. We have demonstrated previously that the lipid composition of liposomal membranes incorporating bilirubin substrate influences the rate of transfer and glucuronidation of bilirubin by hepatic microsomes. To examine the mechanism(s) of substrate transfer, we incorporated radiolabelled bilirubin into small unilamellar model membranes of egg phosphatidylcholine or natural phospholipids in the proportions present in native hepatic microsomes. The rate at which bilirubin was transferred to rat liver microsomes and glucuronidated was then examined in the presence of various endogenous compounds that promote membrane fusion. For bilirubin substrate in membranes of egg phosphatidylcholine, the addition of Ca2+ (2 mM) increased the microsomal glucuronidation rate, whereas retinol enhanced microsomal conjugation rates for bilirubin in membranes of both lipid compositions. When the transfer of [3H]bilirubin from dual-labelled liposomes to microsomes was enhanced by Ca2+ or retinol, there was no associated increase in [14C]phospholipid transfer. Thus it appears likely that bilirubin is transferred to the endoplasmic reticulum by rapid cytosolic diffusion or membrane-membrane collisions, rather than by membrane fusion; this process may be modulated by changes in the lipid microenvironment of the substrate or the effective intracellular concentrations of Ca2+ or retinol. The observation that polymyxin B induced concomitant membrane-membrane transfer of [3H]bilirubin and [14C]phospholipid suggests that under certain circumstances membrane fusion or aggregation may promote the movement of lipophilic substrates in hepatocytes.     

Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2.

Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.     

Oxidation of phenols by cells and cell-free enzymes from Candida tropicalis

A yeast strain isolated from soil by enrichment on phenol as major carbon source was identified as Candida tropicalis. Washed cell suspensions of this strain and cell-free preparations obtained from mechanically disrupted cells oxidized phenol via catechol and cis, cis-muconate. In addition to phenol and the three isomeric diphenols, a number of phenol derivatives, amongst them fluoro-, nitro- and short-chain alkyl-phenols, were oxidized by the organism. However, no significant oxygen uptake could be demonstrated in the presence of pyrogallol, phloroglucinol, the cresols, the m-and p-hydroxy-benzoates, methoxylated phenol derivatives, benzene or toluene. Cell-free preparations from the yeast strain exhibited activity of phenol hydroxylase and of catechol 1,2-oxygenase. Both enzymes appeared in the soluble cell fraction. Both exhibit broad substrate specificities. The relative specific activity of the ring-cleaving enzyme towards various substrates seems to be dependent on the phenolic inducer.     

Influence of para-substituents on the oxidative metabolism of o-nitrophenols by Pseudomonas putida B2

Pseudomonas putida B2 is able to grow on o-nitrophenol (ONP) as the sole source of carbon and nitrogen. ONP was converted by a nitrophenol oxygenase to nitrite and catechol. Catechol was then attacked by a catechol 1,2-dioxygenase and further degraded through an ortho-cleavage pathway. ONP derivatives which were para-substituted with a methyl-, chloro-, carboxy-, formyl- or nitro-group failed to support growth of strain B2. Relevant catabolic enzymes were characterized to analyze why these derivatives were not mineralized. Nitrophenol oxygenase of strain B2 is a soluble, NADPH-dependent enzyme that is stimulated by magnesium, manganese, and calcium ions. It is active toward ONP, 4-methyl-, 4-chloro-, and to a lesser extent, 4-formyl-ONP but not toward 4-carboxy- or 4-nitro-ONP. In addition, 4-formyl-, 4-carboxy-, and 4-nitro-ONP failed to induce the formation of nitrophenol oxygenase. Catechol 1,2-dioxygenase of strain B2 is active toward catechol and 4-methyl-catechol but only poorly active toward chlorinated catechols. 4-Methyl-catechol is likely to be degraded to methyl-lactones, which are often dead-end metabolites in bacteria. Thus, of the compounds tested, only unsubstituted ONP acts as an inducer and substrate for all of the enzymes of a productive catabolic pathway.     

The temporary postnatal decline in glucuronidation of certain phenols by rat liver.

A temporary but marked postnatal decline in UDP-glucuronosyltransferase activity occurs in homogenates and microsomes from rat liver. The profile of this trough and its time of occurrence (maximal over 13-16 days) are almost identical with the two substrates 2-aminophenol and 1-naphthol, whose rates of glucuronidation differ 10-fold. The trough is greatest with digitonin-activated preparations, least with fresh latent ('native') enzyme and intermediate when the native enzyme is treated with its specific activator UDP-N-acetylglucosamine (UDP-GlcNAc). Less detailed evidence supports similar conclusions with 4-nitrophenol as substrate. The trough is not due to the presence of an inhibitor of the transferase in rat liver at 15 days of age. Over the whole perinatal period, including the time of the trough, the enzyme in homogenates can be activated by UDP-GlcNAc; the microsomal enzyme is activated to a rather lesser degree perinatally, and evidence suggests this may be due to artefacts introduced during tissue fractionation. When the overall process of glucuronidation is studied in snips of intact liver offered high concentrations of the two different phenols, the trough is again evident over the same period as observed with broken cells, and of equal depth for both substrates. The infant rat is therefore probably less able to glucuronidate hepatically these phenols over the suckling or early weaning period than are the adult, late foetus or newborn, and may be especially incompetent at 13-16 days of age.     

Stimulation of microsomal uridine diphosphate glucuronyltransferase by glucuronic acid derivatives

The glucuronic acid adducts of 1-naphthol, 2-naphthol and 4-methylumbelliferone activate microsomal UDP-glucuronyltransferase (EC 2.4.1.17) when the enzyme is assayed with p-nitrophenol as aglycone. Phenyl glucuronide and oestriol 3beta-glucuronide also activate UDP-glucuronyltransferase. but to a lesser extent. Activation by glucuronides is not dependent on metal ions, but is blocked by prior treatment of microsomal fractions with p-chloromercuribenzoate. The kinetic mechanism of activation is concluded to be an increase in the affinity of the enzyme for UDP-glucuronic acid. Activation by 1-naphthyl glucuronide, at high concentrations of p-nitrophenol, is not affected by 1-naphthol. Apparently 1-naphthyl glucuronide activates the preparation by binding at a site that is separate from the site of glucuronidation of 1-naphthol. Further evidence for the existence of distinct effector sites for the glucuronides was provided by the finding that activation by glucuronides is inhibited competitively by aglycone glucosides. These glucosides do not inhibit the rate of glucuronidation of p-nitrophenol in the absence of glucuronide adducts, nor do they alter the rate of glucuronidation of 1-naphthol. When UDP-glucuronyltransferase is assayed with 1-naphthol as aglycone it is activated by p-nitrophenyl glucuronide, 4-methyl-umbelliferyl glucuronide and under appropriate conditions by its own glucuronide. These activations are similarly inhibited by aglycone glucosides. p-Nitrophenyl glucuronide also stimulates the rate of glucuronidation of o-aminophenol, o-aminobenzoate and bilirubin.     

Strain differences in purified rat hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.