Bilirubin glucuronyltransferase. Specific assay and kinetic studies

1. Bilirubin glucuronide was synthesized in vitro in a system containing a rat liver microsomal fraction, UDP-glucuronic acid, Mg(2+) and bilirubin. The enzymic synthesis was accomplished without the addition of a bilirubin carrier. 2. Azobilirubin and azobilirubin glucuronide were separated by t.l.c. and paper chromatography and the measurement of the conjugate provided a specific assay for bilirubin UDP-glucuronyltransferase (EC 2.4.1.17). 3. This diazo compound was labelled when [U-(14)C]UDP-glucuronic acid was employed in the transglucuronidation reaction. 4. Identity of the glucuronide nature of the product was further confirmed by hydrolysis with beta-glucuronidase prepared from limpets and Helix pomatia. In each instance azobilirubin and glucuronic acid were liberated. 5. There was a close correlation between the bilirubin glucuronyl-transferase activity as measured by two procedures, colorimetric and radioisotopic. The specific activities so measured were 19nmol of bilirubin ;equivalents' conjugated/h per mg of protein and 16.9-18.4nmol of UDP-glucuronic acid incorporated/h per mg of protein, respectively. On this basis, it was concluded that the major product formed in vitro was bilirubin monoglucuronide; this represents about 77% of the total products formed. 6. The K(m) values for bilirubin and UDP-glucuronic acid at pH8.2 are 3.3x10(-4)m and 1.67x10(-3)m, respectively. 7. The addition of Mg(2+) at a final concentration of 5mm to the reaction mixture increased the rate of conjugation by 5.6-fold in the microsomal preparation that had been subjected to overnight dialysis against 10mm-EDTA (disodium salt). 8. Diethyl-nitrosamine at a final concentration of 1-20mm has no effect on the glucuronidation of bilirubin in vitro.     

An improved assay for steroid glucuronyltransferase in rat liver microsomes

A simple and economical method of assaying rat liver microsomal estrone and testosterone glucuronyltransferase activity has been developed. Liver microsomes were activated by pretreatment with Lubrol WX. The incubation was carried out at 37°C for 30 min and contained 30–600 μm steroid, 1–2 mm UDP-glucuronic acid, 10 mm MgCl₂, and 80–150 μg of microsomal protein. Enzyme activities showed a maximum at pH 8.8 with Tris-HCl buffer. After incubation the unreacted substrate was quantitatively removed by a single extraction with dichloromethane. The glucuronide was estimated by counting an aliquot of the aqueous phase in a liquid scintillation counter. The variation coefficients with estrone and testosterone as substrates were 6.0 and 4.0%, respectively.     

Species differences in the conjugation of 4-hydroxy-3-methoxyphenylethanol with glucuronic acid and sulphuric acid.

The biosynthesis of the glucuronide and sulphate conjugates of 4-hydroxy-3-methoxyphenylethanol was demonstrated in vitro by using the high-speed supernatant and microsomal fractions of liver respectively. These two conjugates were also produced simultaneously by using the post-mitochondrial fraction of rat, rabbit or guinea-pig liver. In contrast only the glucuronide was synthesized by human liver and only the sulphate by mouse and cat livers. Neither of these conjugates was formed by the kidney or the small or large intestine of the rat. A high sulphate-conjugating activity was observed in mouse kidney; the rate of sulphation of 4-hydroxy-3-methoxyphenylethanol with kidney homogenate and high-speed supernatant preparations was 1.8 times greater than with liver preparations. The sulpho-conjugates of 4-hydroxy-3-methoxyphenylethanol and 4-hydroxy-3-methoxy-phenylglycol were also formed by enzyme preparations of rabbit adrenal and rat brain; the glycol was the better substrate in the latter system. Mouse brain did not possess any sulphotransferase activity. For the conjugation of 4-hydroxy-3-methoxyphenylethanol by rabbit liver, the Km for UDP-glucuronic acid was 0.22 mM and that for Na2SO4 was 3.45 mM. The sulphotransferase has a greater affinity for 4-hydroxy-3-methoxyphenyl-ethanol than has glucuronyltransferase, as indicated by their respective Km values of 0.036 and 1.3 mM. It was concluded that sulphate conjugation of 4-hydroxy-3-methoxyphenylethanol predominates in most species of animals.     

The biosynthesis of tyramine glucuronide by liver microsomal fractions.

Labelled tyramine glucuronide was synthesized in vitro from UDP-[14C]glucuronic acid, [14C]tyramine or [3H]tyramine. The glucuronidation was carried out at pH9.2 in the presence of a monoamine oxidase inhibitor, trans-2-phenylcyclopropylamine. The Km values for tyramine were 69 and 125 micrometer and those for UDP-glucuronic acid were 260 and 290 micrometer respectively for guinea-pig and rat liver microsomal preparatons. The specific activities of microsomal glucuronyltransferase measured in fresh hepatic preparations of guinea pig, mouse and rat were respectively 601, 251 and 235 pmol of [14C]tyramine glucuronide/min per mg of protein. Increase in activity ranged from 2- to 6-fold in preparations which were frozen and thawed once and 5.4- to 10-fold when the freezing and thawing was repeated. Rabbit liver has very low activity, and monkey liver and intestine were completely devoid of this conjugating capacity.     

The effect of steroids and nucleotides on solubilized bilirubin uridine diphosphate glucuronyltransferase

1. It was confirmed that bilirubin glucuronyltransferase can be obtained in solubilized form from rat liver microsomes. 2. Michaelis-Menten kinetics were not followed by the enzyme with bilirubin as substrate when the bilirubin/albumin ratio was varied. High concentrations of bilirubin were inhibitory. 3. The K(m) for UDP-glucuronic acid at the optimum bilirubin concentration was 0.46mm. 4. Low concentrations of Ca(2+) were inhibitory in the absence of Mg(2+) but stimulatory in its presence; the converse applied for EDTA. 5. UDP-N-acetylglucosamine and UDP-glucose enhanced conjugation by untreated, but not by solubilized microsomes. 6. The apparent 9.5-fold increase in activity after solubilization was probably due to the absence of UDP-glucuronic acid pyrophosphatase activity in the solubilized preparation. 7. The activation of solubilized enzyme activity by ATP was considered to be a result of chelation of inhibitory metal ions. 8. The solubilized enzyme activity was inhibited by UMP and UDP. The effect of UMP was not competitive with respect to UDP-glucuronic acid. 9. A number of steroids inhibited the solubilized enzyme activity. The competitive effects of stilboestrol, oestrone sulphate and 3beta-hydroxyandrost-5-en-17-one, with respect to UDP-glucuronic acid, may be explained on an allosteric basis.     

Epoxide hydratase assay in human platelets using hepoxilin A3 as a lipid substrate

1-14C-labelled hepoxilin A3 (8-hydroxy-11,12-epoxyeicosa-5,9,14-trienoic acid) was generated from 1-14C-labelled arachidonic acid during incubation with a rat lung preparation lacking epoxide hydratase activity. The HPLC purified hepoxilin A3 gave only two isomeric 8,11,12-triols (termed trioxilins A3) upon incubation with a rat lung preparation containing epoxide hydratase activity. Based on this simple reaction an assay was developed using only 2000 cpm/tube of substrate and aliquots of a homogenate of platelet membranes from man. Products were assayed by thin-layer radiochromatography. Males were noted to have higher epoxide hydratase activity for this substrate than females.     

Simplified spectrophotometric assay for microsomal 3-hydroxy-3-methylglutaryl CoA reductase by measurement of coenzyme A

A new assay for 3-hydroxy-3-methylglutaryl CoA reductase (mevalonate:NADP oxidoreductase [acylating CoA], EC 1.1.1.34) is based upon the measurement of released coenzyme A (SH) during the reduction of 3-hydroxy-3-methylglutaryl CoA to mevalonate. Coenzyme A was measured in the presence of dithiothreitol, required for activity, by reaction with 5,5'-dithiobis(2-nitrobenzoic acid). Sodium arsenite forms a complex with the dithiol, but not with monothiols. Thus, reduced coenzyme A reacts instantaneously with the reagent and dithiothreitol reacts slowly. The absorbance due to the coenzyme A-5,5'-dithiobis(2-nitrobenzoic acid) reaction is determined by extrapolating the linear (dithiol) absorbance-time curve to the time of addition of the reagent. After subtraction of control absorbance (deletion of NADPH), the concentration of CoA-SH is calculated from epsilon(max) = 1.36 x 10(4) at 412 nm. The method of protein removal and reduction of sulfhydryl groups on the enzyme are critical. This method provides an immediate assay. Recovery of reduced coenzyme A was 98.7%. The assay is applicable for microsomes or purified enzyme and has an effective range of 0.5-50 nmoles of coenzyme A. It was applied to kinetic measurement of the pigeon liver microsomal enzyme reaction. The apparent K(m) value for 3-hydroxy-3-methylglutaryl CoA was 1.75 x 10(-5) M, and for NADPH the value was 6.81 x 10(-4) M. This method was compared with the dual-label method at high and low levels of activity. The data were not statistically different.     

A novel glucuronyltransferase in nervous system presumably associated with the biosynthesis of HNK-1 carbohydrate epitope on glycoproteins.

Recently, embryonic chicken brain extract was shown to contain a glucuronyltransferase, which transfers glucuronic acid from UDP-glucuronic acid to glycolipid acceptors (neolactotetraosyl ceramide). The enzyme was also suggested to transfer glucuronic acid to glycoprotein acceptors (asialoorosomucoid) (Das, K. K., Basu, M., Basu, S., Chou, D. K. H., and Jungalwala, F. B. (1991) J. Biol. Chem. 266, 5238-5243). In this study, the glucuronyltransferase activity in rat brain extract was separated into two groups by UDP-glucuronic acid-Sepharose CL-6B column chromatography. The enzyme recovered predominantly in the effluent fraction (GlcAT-L) catalyzed the transfer of glucuronic acid to glycolipid acceptors but not to glycoprotein acceptors, whereas the enzyme recovered in the eluate fraction (GlcAT-P) transferred glucuronic acid most predominantly to glycoprotein acceptors and very little to glycolipid acceptors. GlcAT-P was able to transfer glucuronic acid to oligosaccharide chains on asialoorosomucoid. The enzyme recognized a terminal lactosamine structure, Gal beta 1-4GlcNAc, on glycoproteins. It was localized in the nervous system and was hardly detectable in other tissues, including the thymus, spleen, lung, kidney, and liver. Although GlcAT-L and GlcAT-P shared some properties in common such as tissue distributions and developmental changes, they exhibited marked differences in their phospholipid dependence and in their pH profiles, apart from their respective acceptor preference to glycolipids and glycoproteins. The acceptor specificity and tissue distribution suggest that a novel glucuronyltransferase, GlcAT-P, is involved in the biosynthesis of the sulfoglucuronylgalactose structure in the HNK-1 carbohydrate epitope that is expressed on glycoproteins.     

The submicrosomal distribution of hepatic uridine diphosphate glucuronyltransferases in the rabbit

1. Rabbit liver microsomes were subfractionated into rough- and smooth-surfaced types, and glucuronyltransferase activity in each microsomal subfraction was determined with p-nitrophenol, o-aminophenol and phenolphthalein as substrates. The glucuronyltransferase activity measured with p-nitrophenol and o-aminophenol as substrates was localized predominantly in rough-surfaced microsomes. Glucuronyltransferase measured with phenolphthalein as substrate was equally present in rough- and smooth-surfaced microsomes. 2. Phenobarbital pretreatment of rabbits did not stimulate any of the glucuronyltransferase activities measured in either rough- or smooth-surfaced microsomes. 3. Preincubation of rabbit liver microsomes for 30-60min. at 37 degrees under oxygen did not cause any loss of glucuronyltransferase activity. Such preincubation caused either no change or increased enzyme activity in both submicrosomal fractions. The relative distribution of transferase activity in rough- and smooth-surfaced microsomes was not affected by preincubation.     

Phenol sulphotransferase and uridine diphosphate glucuronyltransferase from rat liver in vivo and in vitro. 2,6-Dichloro-4-nitrophenol as selective inhibitor of sulphation

Microsomal UDP-glucuronyltransferase and cytosolic sulphotransferase share many substrates, such as phenols and hydroxamic acids. In a search for a selective inhibitor of sulphation, several phenolic compounds were tested. 2,6-Dichloro-4-nitrophenol is introduced as a selective inhibitor of sulphation in vivo, having no effect on UDP-glucuronyltransferase activity. As substrate for both conjugating enzymes the phenolic drug harmol (7-hydroxy-1-methyl-9H-pyrido[3,4-b]indole) was used. In the rat in vivo 2,6-dichloro-4-nitrophenol caused almost complete inhibition of harmol sulphation after a single intraperitoneal injection (26mumol/kg) for 48h; the percentage of harmol sulphated decreased from 75% in controls to 5% in the treated rats. The percentage of harmol glucuronidated increased from 25 to 95%. Pentachlorophenol was equally effective but also highly toxic. Salicylamide had only a very-short-lasting inhibitory effect on sulphation. In vitro, 2,6-dichloro-4-nitrophenol inhibited sulphation of harmol by a rat liver postmitochondrial supernatant completely at 1mum, whereas even at 100mum it had no effect on glucuronidation of harmol. It is concluded that 2,6-dichloro-4-nitrophenol is a selective inhibitor of sulphation and, further, that its long duration of action makes it suitable for studies on the regulatory role of sulphation in some biological processes.     

Effects of some antitumor agents on growth and glycolytic enzymes of the flagellate Crithidia

Some antitumor agents known to specifically inhibit certain tumor cell enzymes were examined for activity against glycolytic enzymes and growth of the insect trypanosomatid, Crithidia fasciculata. The cytoplasmic enzymes hexokinase, alpha-glycerophosphate dehydrogenase, malic dehydrogenase, and glucose-6-phosphate dehydrogenase were tested. Agaricic acid (2-hydroxy-1,2,3-nonadecane tricarboxylic acid) was highly inhibitory (50 to 100%) to malic and alpha-glycerophosphate dehydrogenases at approximately 3 x 10(-5)m; 2-(p-hydroxyphenyl)-2-phenylpropane (2 x 10(-4)m), and 5,6-dichloro-2-benzoxazolinone (5 x 10(-4)m) were less effective (50% inhibition) against them. The antiprotozoal agents primaquine (4 x 10(-4)m) and Melarsoprol (8 x 10(-4)m) were 30 to 40% inhibitory. Agaricic acid, 2-(p-hydroxyphenyl)-2-phenylpropane, and 5,6-dichloro-2-benzoxazolinone inhibited growth of Crithidia at less than 10(-4)m. Eight other test compounds from the Cancer Chemotherapy National Service Center (CCNSC) were not toxic to cell growth, although two (4-biphenylcarboxylic acid and 1-[p-chlorobenzyl]-2-ethyl-5-methyl-indole-3-acetic acid) inhibited Crithidia alpha-glycerophosphate dehydrogenase below 1 mm. All of the compounds used specifically inhibited cancer cell alpha-glycerophosphate dehydrogenase. The corresponding enzyme in pathogenic African trypanosomes is important in their terminal respiration. C. fasciculata may be useful in preliminary evaluation of chemotherapeutic agents as potential trypanocides.     

Assay and properties of digitonin-activated bilirubin uridine diphosphate glucuronyltransferase from rat liver

1. The bilirubin UDP-glucuronyltransferase assay described by Van Roy & Heirwegh (1968) has been improved. 2. Extraction of final azo-derivatives is rendered more simple and efficient by thorough emulsification and by cooling. 3. Pretreatment of homogenates and cell fractions with digitonin increases the sensitivity of the assays and gives less variable results than those with untreated preparations. The activation procedure is flexible. 4. Blank values (obtained from incubation mixtures from which activating bivalent metal ion and UDP-glucuronic acid were omitted) are low. No endogenous conjugate formation could be detected except with untreated, fresh liver homogenates. Control incubation mixtures containing the latter preparations are preferably kept at 0 degrees C. 5. With activated microsomal preparations, rates of breakdown of UDP-glucuronic acid (as monitored by release of P(i)) were low. Little if any increase in enzyme activity was found when UDP-N-acetylglucosamine was included in the incubation mixtures. 6. Slight deviation from Michaelis-Menten kinetics with respect to bilirubin observed at low substrate concentrations is probably related to the use of binding protein in the assay mixtures. Michaelis-Menten kinetics were followed with respect to UDP-glucuronic acid. Part of the enzyme in microsomal preparations from rat liver functioned independently of added bivalent metal ions. Mn(2+) was slightly more, and Ca(2+) somewhat less, stimulatory than Mg(2+). The Mg(2+)-dependent fraction showed Michaelis-Menten kinetics with respect to the added Mg(2+). 7. The enzyme activities found were higher than values reported in the literature for untreated or purified preparations from rat liver. They were above reported values of the maximal biliary excretion rate of bilirubin.     

The formation of glucosides of isoflavones and of some other phenols by rabbit liver microsomal fractions

1. Rabbit liver microsomal fractions in vitro effected the transfer of glucuronic acid from UDP-glucuronic acid to biochanin A, formononetin, daidzein, genistein and equol. Only monoglucuronides were formed. 2. The same isoflavones were converted into monoglucosides when UDP-[6-(3)H]glucose was substituted for UDP-glucuronic acid in the incubation medium in vitro. The glucosides were formed in much lesser yield than were the glucuronides. 3. The glucoside of genistein was identified as genistin (genistein 7-glucoside) by Sephadex chromatography and reverse isotope dilution. 4. The specificity of the glucuronyl- and glucosyl-transfer mechanisms was compared for a series of steroids and other phenols in addition to the isoflavones. It was concluded that separate transferases were responsible for the formation of the two types of glycosides.     

Perinatal developmental changes in hepatic UDP-glucuronyltransferase.

Postnatal developmental changes in hapatic microsomal UDP-glucuronyltransferase were studied in the rat. The previously reported postnatal decline in the capacity of microsomal fractions to glucuronidate p-nitrophenol was found to be observable in unperturbed preparations only at non-saturating concentrations of the substrate UDP-glucuronic acid. At saturating concentrations of UDP-glucuronic acid, activity is identical in newborns and adults. Kinetic analysis revealed that the enzyme from liver of newborns has a much higher affinity for UDP-glucuronic acid than does the enzyme in adults, but the same activity at Vmax. On the other hand, the enzyme from adult liver microsomal fractions can be activated by the physiological allosteric effector UDP-N-acetylglucosamine, whereas the enzyme from newborns is largely unaffected by it. Thus it appears that the number of enzyme active sites is not changing; rather, the enzyme is maturing to a more highly regulable form. There were also differences between the enzymes in newborns and adults in their response to perturbation of the membrane-lipid environment by detergent and phospholipase A. Possible interpretations of these differences are discussed.     

Effect of atractyloside on glucose and pyruvate metabolism in rat diaphragm muscle

1. In the presence of 1.2mm-atractyloside oxygen uptake by rat diaphragm muscle incubated with 5.6mm-glucose decreases, as well as glycogen synthesis and carbon dioxide production. Lactate formation from glucose increases, but that of phosphoglycerate diminishes fivefold. 2. When pyruvate is used as substrate, atractyloside decreases oxygen uptake. 3. The specific radioactivity of the (14)CO(2) (mumoles of (14)CO(2)/mumole of oxygen), calculated at concentrations of [1-(14)C]pyruvate between 0.091mm and 91mm, lies between 3.1x10(-4) and 5.7x10(-1). Atractyloside increases the specific radioactivity of the (14)CO(2) with the lowest concentrations of substrate and has no effect when the substrate concentration is 91mm. 4. No appreciable effect of atractyloside on the anaerobic production of (14)CO(2) from [1-(14)C]pyruvate at various incubation times and various concentrations is found. 5. It is suggested that atractyloside induces anaerobic conditions in the tissue. Further, it produces a rise in the pyruvate concentration and an ATP deficiency in the cell. Consequently it stimulates pyruvate dismutation, and glycolysis, to which phosphorylation is linked at the substrate level.     

Optimal assay conditions for liver UDP-glucuronosyltransferase from the rainbow trout, Salmo gairdneri

Several kinetic characteristics and assay dependence of UDP-glucuronosyltransferase were studied with microsomal preparations made from liver of rainbow trout. The optimal enzyme assay, performed by incubating less than 5 mg microsomal protein/ml assay buffer for 20 min at 25 degrees C and in pH 7.0, contains 2.5 X 10(-5) M p-nitrophenol (p-NP) and 2.5 X 10(-3) M UDP-glucuronic acid (UDPGA). Apparent Km values revealed that the affinity of trout enzyme for p-NP and UDPGA is, respectively, about 70 and 10 times higher than that of rat enzyme. The optimized method will be used for aquatic bioassays, e.g. when assessing the influencing of toxic effluents from the pulp and paper industry.     

The inhibition of cytoplasmic acetoacetyl-CoA thiolase by a triyne carbonate (L-660, 631)

The compound L-660, 631 (2-oxo-5-(1-hydroxy-2,4,6-heptatriynyl)-1,3-dioxolane-4 heptanoic acid), a natural product isolated from an Actinomycete culture, was found to inhibit rat liver cytosolic acetoacetyl-CoA thiolase, the first step in the cholesterol biosynthesis pathway, with an IC50 of 1.0 x 10(-8) M. The inhibitor had no effect on other sulfhydryl containing enzymes of lipid synthesis such as HMG-CoA synthase, HMG-CoA reductase, and fatty acid synthase. When tested in cultured human liver Hep G2 cells the compound inhibited the incorporation of 14C-acetate and 14C-octanoate into sterols 56% and 48% respectively at 3 x 10(-6) M with no effect on fatty acid synthesis. No noticeable effect was seen on fatty acid biosynthesis. This strongly suggests that the locus of inhibition of acetate incorporation into sterols found with this compound is the acetoacetyl-CoA thiolase step in the cholesterol biosynthesis pathway.     

Further purification and characterization of the acid α-glucosidase

1. Centrifugation of rat liver acid glucosidase, which had been purified by adsorption on dextran gel, on a density gradient of sucrose showed the enzyme to be impure. 2. Preliminary purification of the enzyme before the gel filtration improved the final degree of purity of this preparation. Disc gel electrophoresis of this preparation showed a single band of protein. 3. The sedimentation co-efficient and the molecular weight determined on a sucrose gradient were 4.9-5.1s and 76000-83000 respectively for the rat liver enzyme, and 5.6s and 97000 for the acid alpha-glucosidase purified by means of the same procedure from the human kidney. 4. The Michaelis constants of rat liver and human kidney enzyme were 4.7x10(-3)m and 13.6x10(-3)m respectively with maltose as substrate. 5. The enzyme from both tissues was inhibited by tris and by erythritol. The inhibition of the rat liver acid glucosidase by erythritol was competitive.     

Investigation of rat liver microsomal 6 beta-hydroxylation of 4-androstene-3,17-dione and 4-pregnene-3,20-dione using methodology which excludes steroid-3-imine induced introduction of the 6 beta-hydroxyl group

To avoid artefactual 6 beta-hydroxylation of 3-oxo-4-ene steroids due to steroid-3-imine formation and rearrangement a combined extraction and liquid chromatography purification procedure for incubated rat liver microsomes has been worked out. With this procedure no nonenzymatic 6 beta-hydroxylation could be observed. Conventional termination of incubations with male rat liver microsomes (105,000 g sediments) and 4-14 C-labelled 4-androstene-3,17-dione (or progesterone) by solvent extraction and evaporation might lead to a 30% overestimation of the formation of 6 beta-hydroxy-derivatives at substrate saturation. Furthermore this work-up procedure produces 6-oxo-derivatives which were not observed when the new procedure was used. By elimination of the artefactual 6-oxygenation some properties of the male rat liver microsomal 4-androstene-3,17-dione 6 beta-hydroxylase have been studied, and the activity has been compared to the artefact produced by solvent extraction and evaporation. Using the combined extraction-liquid chromatography purification it was demonstrated that the microsomal 6 beta-hydroxylase active on 4-androstene-3,17-dione and progesterone was inhibited to 95% by carbon monoxide. This makes previous suggestions regarding participation of a non cytochrome P450-dependent 4-androstene-3,17-dione 6 beta-hydroxylase less likely.     

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.