Properties of membrane-bound bilirubin UDP-glucuronyltransferase in rough and smooth endoplasmic reticulum and in the nuclear envelope from rat liver.

We examined regulatory properties of bilirubin UDP-glucuronyltransferase in sealed RER (rough endoplasmic reticulum)- and SER (smooth endoplasmic reticulum)-enriched microsomes (microsomal fractions), as well as in nuclear envelope from rat liver. Purity of membrane fractions was verified by electron microscopy and marker studies. Intactness of RER and SER vesicles was ascertained by a high degree of latency of the lumenal marker mannose-6-phosphatase. No major differences in the stimulation of UDP-glucuronyltransferase by detergent or by the presumed physiological activator, UDPGlcNAc, were observed between total microsomes and RER- or SER-enriched microsomes. Isolated nuclear envelopes were present as a partially disrupted membrane system, with approx. 50% loss of mannose-6-phosphatase latency. The nuclear transferase had lost its latency to a similar extent, and the enzyme failed to respond to UDPGlcNAc. Our results underscore the necessity to include data on the integrity of the membrane permeability barrier when reporting regulatory properties of UDP-glucuronyltransferase in different membrane preparations.     

Endogenous esterification of bilirubin by liver microsomes. Evidence for an intramicrosomal pool of UDP-glucose and lumenal orientation of bilirubin UDP-glycosyltransferase

Conjugation of natural bilirubin (BR) depends on a hepatic microsomal UDP-glycosyltransferase using UDP-Glc, UDP-xylose, and predominantly UDP-GlcA. We found that esterification of BR occurred when washed intact microsomes derived from rat or guinea pig liver were incubated with BR in the absence of added UDP-sugar. This endogenous esterification was shown to lead predominantly to formation of the two positional isomers of BR monoglucoside and displayed the same regioselectivity as found for the BR monoglucosides formed by microsomes incubated with a saturating concentration of added UDP-Glc. This finding and absence of endogenous esterification in liver microsomes from mutant rats lacking BR UDP-glycosyltransferase activities demonstrated that endogenous esterification depended on UDP-glycosyltransferase and indicated, therefore, that UDP-Glc was present in the intact microsomal vesicles. With UDP-Glc added to the extramicrosomal incubation medium, BR glucosidation was markedly enhanced when the membrane permeability barrier was disrupted by pretreatment of the microsomes with detergent, sonication, or Staphylococcus aureus alpha-toxin. In contrast, such membrane disruption resulted in abolishment of endogenous esterification of BR, and a direct relationship was found between impairment of endogenous esterification and degree of vesicle disruption, suggesting that the UDP-Glc on which endogenous esterification depended was present in the lumenal space of the microsomes. Kinetic evidence and absence of an effect of increasing the microsomal concentration of dolichol-P-Glc (Dol-P-Glc) on endogenous esterification excluded direct or indirect involvement of Dol-P-Glc in the endogenous esterification reaction. Preincubation of intact microsomes with UDP-Glc or UDP-xylose at 37 degrees C, but not at 0 degrees C, led to expansion of the microsomal UDP-sugar pool on which endogenous esterification depended, suggesting that both UDP-sugars can enter the microsomal vesicles by a temperature-dependent mechanism. In contrast to these findings, no increase of BR esterification was detected when the microsomes had been preincubated at 37 degrees C with UDP-GlcA. We conclude that native, intact microsomes contain a lumenal pool of endogenous UDP-Glc and that BR UDP-glucosyltransferase and UDP-xylosyltransferase, by virtue of a lumenal orientation, have direct access to the postulated intramicrosomal pool of nucleotide sugar.     

Bilirubin mono- and di-glucuronide formation by purified rat liver microsomal bilirubin UDP-glucuronyltransferase.

Highly purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver, when reconstituted with Gunn-rat liver microsomes (microsomal fraction), was able to catalyse the conversion of unesterified bilirubin into both bilirubin monoglucuronide and diglucuronide. Under zero-order kinetic conditions for monoglucuronide formation, the fraction of bilirubin diglucuronide formed by incubation of bilirubin with the reconstituted highly purified transferase accounted for 18% of total bilirubin glucuronides, which was only slightly lower than the fraction of diglucuronides (23% of total bilirubin glucuronides) formed by incubation with hepatic microsomes in the presence of UDP-N-acetylglucosamine or Lubrol. The reconstituted purified enzyme also catalysed the UDP-glucuronic acid-dependent conversion of bilirubin monoglucuronide into diglucuronide and, when bilirubin was incubated with UDP-glucose or UDP-xylose, the formation of bilirubin glucosides and xylosides respectively. These results suggest that a single microsomal bilirubin UDP-glycosyltransferase may be responsible for the formation of bilirubin mono- and di-glycosides.     

Identification of increased amounts of UDP-glucuronyltransferase protein in phenobarbital-treated chick-embryo liver cells.

UDP-glucuronyltransferase activity of neonatal-chick liver or phenobarbital-treated chick-embryo liver catalysed the glucuronidation of 1-naphthol, 4-nitrophenol and 2-aminophenol. Only low transferase activity towards testosterone was detected, and activity towards bilirubin was not detectable. Liver microsomal transferase activity towards the three phenols was increased approx. 20-50-fold by phenobarbital treatment of chick embryos or by transfer of liver cells into tissue culture. A single form of UDP-glucuronyltransferase, which appears to catalyse the glucuronidation of these three phenols, was purified to near homogeneity from phenobarbital-treated chick-embryo liver microsomal fraction for the first time. The use of this purified enzyme as a standard protein facilitated the identification of this protein in chick-embryo liver microsomal fraction. Further, the accumulation of this microsomal protein was observed following phenobarbital treatment of chick embryos and during tissue culture of chick-embryo liver cells. The value of this model system for the study of the induction of UDP-glucuronyltransferase by drugs and hormones is discussed.     

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.     

Isolation of an activator of bilirubin glucuronyltransferase from normal and jaundiced Gunn rats

A microsomal activator of the UDP-glucuronyltransferase for bilirubin has been isolated from lubrol solubilized and salt fractionated liver microsomes. The activator has been partially purified by anion exchange and molecular sieving chromatography and found to have a molecular weight of about 60 kDa. The activator is present in liver from normal and bilirubin UDP-glucuronyltransferase deficient Gunn rats. When tested with purified UDP-glucuronyltransferase for bilirubin it accelerated the conjugation rate 10 fold but with the purified UDP-paranitrophenol transferase the rate of conjugation was increased only 1.5 times.     

Hepatic microsomal glucuronidation of bilirubin in unilamellar liposomal membranes. Implications for intracellular transport of lipophilic substrates

It has been assumed that following hepatic uptake, bilirubin is bound exclusively to cytosolic proteins prior to conjugation by microsomal UDP-glucuronyl-transferase. Since bilirubin partitions into lipid rather than the aqueous phase at neutral pH, we postulated that bilirubin reaches the sites of glucuronidation by rapid diffusion within membranes. To examine this hypothesis, [14C]bilirubin was incorporated into the membrane bilayer of small unilamellar liposomes of egg phosphatidylcholine. Radiochemical assay of this membrane-bound substrate in a physiologic concentration, using native rat liver microsomes, demonstrated immediate formation of bilirubin glucuronides at a more rapid initial velocity than for bilirubin bound to the high-affinity sites of purified cytosolic binding proteins, i.e. glutathione S-transferases (p less than 0.025) or native liver cytosol (p less than 0.05). Kinetic analysis suggested that the mechanisms of substrate transfer from liposomal membranes and from purified glutathione S-transferases to microsomal UDP-glucuronyltransferase were similar. The exchange of 3H- and 14C-labeled bilirubin substrate between binding proteins and liposomal membranes was then investigated using Sepharose 4B chromatography. As the concentration of bilirubin was increased relative to that of protein, net transfer of substrate from the protein to the membrane pool was observed. These findings indicate that bilirubin is efficiently transported by membrane-membrane transfer to hepatic microsomes, where it undergoes rapid conjugation. Bilirubin entering hepatocytes may partition between membrane and cytosolic protein pools, but as intracellular bilirubin concentration increases, the membrane pool is likely to provide a greater proportion of the substrate for glucuronidation.     

Stimulation by secretin of bilirubin UDP-glycosyltransferase activities and of cytochrome P-450 concentration in rat liver.

The activity of bilirubin UDP-glucuronyltransferase in liver tissue was increased 1.5-fold after 90 min of secretion administration (4 i.u./h per kg body wt.) in anaesthetized Wistar rats biopsied half-hourly over a period of 2 h. In unanaesthetized R/A Wistar rats, activities of liver enzymes were assayed after administration secretin for 1 h. Bilirubin UDP-glycosyltransferase activities and cytochrome P-450 concentration were increased, but p-nitrophenol UDP-glucuronyltransferase and UDP-glucose dehydrogenase activities remained unchanged.     

Hepatic microsomal glucuronidation of bilirubin is modulated by the lipid microenvironment of membrane-bound substrate

Hepatocyte intracellular membranes may facilitate the directed movement of bilirubin and other hydrophobic substrates to the active site of UDP-glucuronyltransferase in the endoplasmic reticulum. We postulated that the lipid composition and physical properties of membranes that transport substrate may modulate bilirubin glucuronidation. To examine this hypothesis, we incorporated [14C]bilirubin substrate into the membrane bilayer of small unilamellar liposomes composed of native phospholipid purified from rat hepatic microsomes. The initial velocity of bilirubin glucuronide formation in rat liver microsomes, measured by radiochemical assay, was considerably more rapid than for bilirubin in liposomes of egg phosphatidylcholine (p less than 0.001). Moreover, the ratio of bilirubin diglucuronide to monoglucuronides synthesized was markedly increased (p less than 0.01), approaching that observed in normal rat bile. Although the rates of bilirubin glucuronidation did not correlate with fluidity of the liposomal membrane core region, specific phospholipid head groups were associated with an increase, and cholesterol a decrease, in rates of glucuronidation. Movement of [3H]bilirubin from dual-labeled liposomes to microsomes occurred without concomitant [14C]phospholipid transfer. Thus, the lipid composition of membranes incorporating bilirubin appears to modulate the rate of glucuronidation and the relative rates of bilirubin mono- and diglucuronide formation. Phospholipid head groups on the surface of the bilayer, not the hydrocarbon core regions, may be implicated in the rapid process of membrane transport, which is likely to involve membrane-membrane collisions or diffusion of free substrate rather than membrane fusion.     

In vitro drug metabolism in male and female athymic,nude mice

Drug metabolism was studied in hepatic microsomal and post microsomal supernatant fractions from male and female athymic nude mice (nu/nu) and heterozygous (+/nu) and homozygous (+/+) wild-type controls. In males, the following enzyme activities were higher in athymic mice than in the wild-type: NADPH cytochrome c reductase, ethylmorphine and aminopyrine N-demethylases, native UDP glucuronyltransferase, and glutathione (GSH) S-aryltransferase. No differences were observed between groups in UDPNAG-activated UDP-glucuronyltransferase, N-acetyltransferase, or aniline hydroxylase activities or in amounts of cytochrome P-450. In female athymic mice, only ethylmorphine and aminopyrine N-demethylase activities were significantly higher than in female wild-type controls (+/+). The female athymic mice had mixed function oxidase activities that were less than the male athymic mice. There were no sex or strain differences in response to treatment with phenobarbital or 3-methylcholanthrene.     

Reversible ATP-induced inactivation of branched-chain 2-oxo acid dehydrogenase.

1. Antiserum was raised against purified Wistar-rat liver UDP-glucuronyltransferase. 2. UDP-glucuronyltransferase activities towards 4-nitrophenol, bilirubin, 1-naphthol and morphine were co-immunoprecipitated from solubilized Wistar-rat liver preparations. 3. UDP-glucuronyltransferase activities towards 1-naphthol, 2-aminophenol and 4-nitrophenol were precipitated from solubilized Gunn-rat liver preparations by this antiserum. 4. UDP-glucuronyltransferase activities towards 1-naphthol, 4-nitrophenol and bilirubin, from Wistar-rat liver, were slightly inhibited by antiserum, whereas 1-naphthol UDP-glucuronyltransferase activity from Gunn-rat livers was greatly inhibited. 5. Measurable Wistar-rat liver glucuronyltransferase activities in washed immunoprecipitates indicate that the enzyme(s) were not merely inhibited by antiserum. 6. Immunoglobulin G purified from this antiserum immunoprecipitated transferase activities towards 4-nitrophenol, bilirubin and 1-naphthol. 7. The washed immunoprecipitates from both rat strains, containing UDP-glucuronyltransferase activity, appear to be similar when analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 8. Radial-immunodiffusion studies suggest that a smaller amount of UDP-glucuronyltransferase protein is present in Gunn-rat liver than in Wistar-rat liver. 9. The significance of these results in relation to the genetic deficiency in the Gunn rat is discussed.     

The influence of fatty acid unsaturation and physical properties of microsomal membrane phospholipids on UDP-glucuronyltransferase activity.

The relationship between lipid composition, the physical properties of microsomal phospholipids and the kinetics of liver UDP-glucuronyltransferase was studied in microsomes from guinea pigs supplied with a normal or a fat-free diet for 28 days. Fatty acid deficiency did not modify either the cholesterol/phospholipid molar ratio or the polar head group composition, but exclusively redistributed the unsaturated fatty acid pattern, by partially exchanging oleic for linoleic acid. This phenomenon accounts for the decrease of both rotational and translational mobilities of the fluorescent probes 1,6-diphenyl-1,3,5-hexatriene (DPH) and pyrene respectively. When the thermotropic behaviour of the different systems was assessed, no transition temperature (gel-liquid-crystalline) between 10 and 40 degrees C was seen as a consequence of the lower degree of unsaturation, either in the microsomal membranes or in the total lipid or total phospholipid extracts from the treated animals. In spite of this, the polarization ratio of trans-parinaric acid and the fluorescence intensity of merocyanine 540 revealed that a significant lateral phase separation occurred at 20-22 degrees C in the extracted phospholipids, which was smoother in the total lipid fractions and in the native microsomal membranes. Fatty acid deficiency caused an upward shift of the midpoint temperature of the lateral phase separation. Furthermore, the phosphatidylcholine extracted from the 'normal' microsomes showed a lateral phase separation centred at a lower temperature than that extracted from 'fat-deficient' microsomes. In contrast, the Arrhenius plot of UDP-glucuronyltransferase from 'normal' microsomes exhibited a change in slope at a higher temperature than that from treated microsomes. These results would suggest that fatty acid deficiency in guinea-pig liver microsomes, while rigidizing the bulk lipids, would segregate the most unsaturated phosphatidylcholine molecules towards the UDP-glucuronyltransferase microenvironment, in accordance with our previous results with cholesterol incorporation [Castuma & Brenner (1986) Biochemistry 25, 4733-4738].     

Analysis of the interaction uridin 5'-diphosphoglucuronic acid with intestinal bilirubin UDP-glucuronyltransferase.

1. Bilirubin UDP-glucuronyltransferase Michaelis-Menten kinetic parameters for UDP-glucuronic acid were studied using native and digitonin activated microsomes obtained from rat intestinal mucosa. 2. The intestinal enzyme showed a lower apparent Vmax compared with the hepatic enzyme in both native and activated microsomes; digitonin pretreatment enhanced Vmax 4 times in the former enzyme and 2 times in the latter. 3. The affinity of UDP-glucuronic acid for the intestinal enzyme was about 2 times lower than that for the hepatic enzyme and it was not substantially modified by detergent neither in the intestine nor in the liver. 4. The lipid analysis of intestinal and hepatic microsomes showed that the former present a higher content of cholesterol and a lower phosphatidylcholine/sphingomyelin ratio than the latter, accordingly the estimation of membrane fluidity using the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicated that intestinal microsomes are more "rigid" than the hepatic ones. 5. These characteristics would provoke a restrictive milieu surrounding the enzyme that modifies its kinetic properties thus limiting its participation in the metabolism of bilirubin.     

Characterization of electron transport enzymes in the envelope of rat liver nuclei.

Nuclei and microsomes were prepared from the livers of normal, phenobarbital (PB)-treated and beta-naphthoflavone (beta-NF)-treated rats, and the contents of several enzymes in both subcellular fractions were examined. In normal rats, the enzyme activities in the nuclear fraction were about one-third of those of microsomes on a phospholipid basis. The induction of some particular enzymes by the drugs was observed with nuclei as well as with microsomes. Cytochrome P-450 and NADPH-cytochrome c reductase were increased by PB treatment and cytochrome P-448 was induced by beta-NF treatment both in nuclei and in microsomes. The extents of inhibition of nuclear enzyme activities by the antibodies against corresponding microsomal enzymes were almost the same as those of the microsomal activities. It was concluded that a microsomal type electron transport system exists in rat liver nuclei, and that nuclear drug-oxidizing activities are inducible by PB or beta-NF as their microsomal counterparts are.     

Characteristics of renal UDP-glucuronyltransferase

Rat renal microsomes catalyzed the glucuronidation of l-naphthol, 4-methylumbelliferone and p-nitrophenol, whereas morphine and testosterone conjugation were not detected. In contrast, all five substrates were conjugated by hepatic microsomes; the activity was typically 5-10 times greater than with renal microsomes. Renal microsomal UDP-glucuronyltransferase toward l-naphthol was fully activated (six-fold) by 0.03% deoxycholate while the hepatic enzyme was fully activated (eight-fold) by 0.05% deoxycholate. Full activation of hepatic UDP-glucuronyltransferase occurred when microsomes had been preincubated at 0 C with deoxycholate for 20 min. This effect of preincubation was not observed with renal microsomes. The presence of 0.25M sucrose in the buffers during renal microsomal preparation resulted in a two-fold greater rate of l-naphthol conjugation in both unactivated and activated microsomes than renal microsomes prepared in phosphate buffers alone. Preparation of hepatic microsomes with or without 0.25M sucrose had no effect on UDP-glucuronyltransferase activity. Unactivated (-deoxycholate) renal enzyme was activated when incubations were done at a low pH (5.7), whereas fully activated (0.03% deoxycholate) renal microsomal UDP-glucuronyltransferase displayed a pH optimum at 6.5. Renal microsomal UDP-glucuronyltransferase activity toward l-naphthol, p-nitrophenol and 4-methylumbelliferone was induced by pretreatment of rats with beta-naphthoflavone and trans-stilbene oxide but not by phenobarbital or 3-methylcholanthrene. These data demonstrate that renal UDP-glucuronyltransferases are different from the hepatic enzymes with regard to biochemical properties, substrate specificity and in response to chemical inducers of xenobiotic metabolism.     

Reconstitution of purified Wistar rat liver bilirubin UDP-glucuronyltransferase into Gunn-rat liver microsomes.

1. Reconstitution of purified bilirubin UDP-glucuronyltransferase from Wistar-rat liver into Gunn-rat liver microsomes provides a better environment than phosphatidylcholine liposomes, such that the final specific activity of the Wistar-rat liver enzyme was increased up to 85 units/mg of protein. 2. Gunn- and Wistar-rat liver microsomes were equally effective for reconstitution of the purified enzyme. 3. The transferase activity does not appear to be fully expressed in the more rigid environment of foetal Wistar-rat liver microsomes. 4. These reconstitution experiments reveal a final specific activity for the purified bilirubin UDP-glucuronyltransferase consistent with the capacity of the whole rat liver to glucuronidate bilirubin and indicate that the absence of this enzyme activity in Gunn-rat liver microsomes is not due to an abnormal microenvironment.     

Electroimmunochemical quantification of UDP-glucuronosyltransferase in rat liver microsomes

Microsomal UDPglucuronosyltransferase(1-naphthol), an enzyme form previously shown to be selectively inducible in rat liver by 3-methylcholanthrene-type inducers, was purified to apparent homogeneity. Rabbit antibodies against this enzyme form precipitated UDPglucuronosyltransferase activities towards 1-naphthol and 4-methylumbelliferone faster and to greater extents than enzyme activities towards bilirubin, oestrone and 4-hydroxybiphenyl. Ouchterlony double-diffusion analysis showed immunochemical similarity of the rat liver enzyme with the enzymes from other organs of the rat (kidney, testes) and the mouse liver but not with the enzyme from cat and human liver. Electroimmunochemical quantification of the enzyme indicated that its level was enhanced 1.3-fold and 2.5-fold in liver microsomes from phenobarbital-treated and 3-methylcholanthrene-treated rats, respectively. The results indicate that 3-methylcholanthrene treatment increases the enzyme level of rat liver microsomal UDPglucuronosyltransferase(1-naphthol). Despite phospholipid-dependence of its catalytic activity microsomal enzyme activity appears to be a good index of the enzyme level.     

Enzymatic conversion of bilirubin monoglucuronide to diglucuronide by rat liver plasma membranes.

Formation of bilirubin monoglucuronide from unconjugated bilirubin requires a microsomal enzyme, UDP-glucuronate glucuronyltransferase (EC 2.4.1.17). Conversion of bilirubin monoglucuronide to bilirubin diglucuronide, the major bilirubin conjugate in bile, was studied in subcellular fractions of rat liver. The highest specific activity for bilirubin diglucuronide formation occurred in a fraction highly enriched in plasma membranes. Studies of reaction stoichiometry and utilization of UDP-D-[14C]glucuronic acid revealed that conversion of bilirubin monoglucuronide to bilirubin diglucuronide is not catalyzed by UDP-glucuronyltransferase, and results from transglucuronidation of bilirubin monoglucuronide, with formation of bilirubin diglucuronide and unconjugated bilirubin. When unconjugated bilirubin was infused intravenously into rats at rates exceeding the maximal hepatic excretory capacity, bilirubin monoglucuronide accumulated in serum and bilirubin diglucuronide was found exclusively in bile as the predominant bilirubin metabolite. These results suggest that formation of bilirubin diglucuronide occurs at the surface membrane of the liver cell. Conversion of bilirubin monoglucuronide to bilirubin diglucuronide may play a role in the transport of bilirubin glucuronides from liver to bile.     

Comparison of hepatic, renal and intestinal bilirubin UDP-glucuronyl transferase activities in rat microsomes

1. Bilirubin UDP-glucuronyltransferase activity and its dependence on substrate concentrations in rat liver, renal cortex and intestinal mucosa microsomes were studied. 2. Bilirubin monoglucuronide synthesis from unconjugated bilirubin was a higher capacity, lower affinity step in comparison with bilirubin diglucuronide formation in the three tissues tested. 3. Bilirubin glucuronide formation in liver microsomes showed a higher capacity but a lower affinity than extrahepatic ones. Renal cortex and intestinal mucosa exhibited similar kinetics parameters. 4. In vitro bilirubin glucuronidation in renal cortex and intestinal mucosa was quantitatively important as compared with the hepatic one.     

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.