Peter I. Mackenzie

A sensitive kinetic assay for UDPGlucuronosyltransferase using l-naphthol as substrate

Abstract A sensitive, kinetic assay for UDPglucuronosyltransferase has been devised using 1-naphthol as substrate. It is based on the continuous fluorometric monitoring of 1-naphthol glucuronide production in the cuvette during the reaction. Interference in the measurement of glucuronide fluorescence by 1-naphthol is negligible, since both aglycone and glucuronide have widely differing fluorescence characteristics in the reaction mixture. This method is as sensitive as other assays using 1-naphthol as substrate but has the added advantage that no extraction procedures are involved and the course of the enzymic reaction can be followed directly.

Cleavage of nascent UDP glucuronosyltransferase from rat liver by dog pancreatic microsomes

Antibody to mouse UDP glucuronosyltransferase, previously shown to cross-react with rat transferase [1], immunoadsorbed 3 electrophoretically distinct transferase forms from the microsomes of untreated and phenobarbital-treated rats and 4 forms from 3-methylcholanthrene treated animals. The forms from phenobarbital-treated or control animals ranged in molecular weights from 49,000 to 52,000 daltons, and those from 3-methylcholanthrene-treated rats ranged from 51,000 to 57,000 daltons. The intensity of the electrophoretic bands indicated that the levels of at least two forms were increased by the administration of either compound. In contrast, only a 52,000-dalton electrophoretic band was observed after immunoadsorption of in vitro translated products using poly(A) RNA isolated from either control, phenobarbital-, or 3-methylcholanthrene-treated rats. When dog pancreatic microsomes were included in the in vitro translation assay for either of the poly(A) RNA preparations, part of the 52,000-dalton band remained and a new band of about 50,000 daltons was generated. This processed transferase form(s) appeared to be inserted into or sequestered by the microsomes. These results indicate that some of the electrophoretic variants of rat liver transferase arise by posttranslational modifications and that at least one rat transferase form undergoes proteolytic cleavage of an approximate 2,000-dalton peptide fragment during insertion into the membrane.

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 .

Differences in udp-glucuronosyltransferase activities in congenic inbred rats homozygous and heterozygous for the jaundice locus

The genic transfer of the jaundice locus (jj) from the Gunn rat into the inbred RHA/++ rat produced congenic inbred homozygous RHA/jj rats which lacked detectable bilirubin UDP-glucuronosyltransferase activity. Congenic inbred RHA/j+ rats contained half the activity for bilirubin of the RHA/++ strain. Constitutive activities for glucuronidation of sixteen substrates of twenty-one tested were inherited additively. Approximately seven groups were discernible based on the defect in activity for these substrates in the RHA/jj strain. Activity for 1-hydroxybenzo[a]pyrene was, after that for bilirubin, the most severely reduced (188-fold), while no differences in the glucuronidation of three androgens and of the 6-hydroxy-, 10-hydroxy-, and 11-hydroxybenzo[a]pyrenes were observed. The conjugation of other substrates was affected to an intermediate extent. Most of the twenty-one glucuronidating activities were induced by phenobarbital in the RHA/jj strain as well as in the RHA/++ and RHA/j+ strains. Activities for 9-hydroxybenzo[a]pyrene and for the 2-hydroxy- and 4-hydroxybiphenyls were induced such that the defect was overcome, and the RHA/jj had the same level of activity as the RHA/++ strain. Cytochrome p-450 content and cytochrome c reductase and aminopyrine demethylase activities were unaffected in the congenic strains. Cytochrome p-450 content and cytochrome c reductase activity were induced approximately 2.5- and 2.0-fold, respectively, by phenobarbital while aminopyrine demethylase activity was induced about 30% in each strain. The congenic inbred rats should provide a stable and reproducible genetic model for studying defective UDP-glucuronosyltransferase specified by the jaundice (jj) locus.

Cell-free translation of mouse liver mRNA coding for two forms of UDP glucuronosyltransferase.

Mouse liver poly(A) RNA, when translated in vitro, produced two forms of UDP glucuronosyltransferase with molecular weights of approximately 50,000 and 54,000 (designated GTm1 and GTm2, respectively). These forms were identified by antibody prepared against GTm1. The mRNA coding for GTm1 was preferentially increased twofold after treatment of mice with 3-methylcholanthrene, while GTm2 mRNA was unaffected. Phenobarbital, however, increased the translatable levels of the mRNAs coding for both proteins approximately twofold. GTm1 was shown to be glycosylated during translation in the presence of dog pancreatic microsomes. This was reflected by a decrease in mobility of the protein in sodium dodecyl sulfate-polyacrylamide gels as compared to GTm1 translated in the absence of microsomes. Further evidence for glycosylation in vivo was demonstrated by an increase in the mobility of GTm1 immunoadsorbed from microsomes treated with endoglycosidase H. In contrast, GTm2 was unmodified. This apparent difference in the state of glycosylation may reflect a difference in the transmembrane distribution of these two enzyme forms, and hence play an important role in determining the type of aglycone glucuronidated and its route of removal from the cell.

Separation of different UDP glucuronosyltransferase activities according to charge heterogeneity by chromatofocusing using mouse liver microsomes: Three major types of aglycones☆

Hepatic UDP glucuronosyltransferase (EC 2.4.1.17) (GT) enzymes in control, phenobarbital- and 3-methylcholanthrene-induced microsomes from C57BL/6N mice have been fractionated according to charge heterogeneity on a chromatofocusing system using a pH 9.5 to 6 gradient. Transferase activities for eleven different substrates were determined on column fractions. Activities toward 3-hydroxybenzo[a]pyrene, phenolphthalein and estrone (type 1 substrates) were enhanced by both effector compounds and always eluted primarily at pH 8.5. In control and phenobarbital-induced microsomes, activities toward testosterone, 4-hydroxybiphenyl, morphine, naphthol and 9-hydroxybenzo[a]pyrene (type 2 substrates) eluted primarily at about pH 6.7. Activities toward p-nitrophenol, 4-methylumbelliferone and 2-hydroxybiphenyl (type 3 substrates) in control and phenobarbital-induced microsomes exhibited two peaks which eluted at pH 8.5 and 6.7. 3-Methylcholanthrene treatment increased almost exclusively activities which eluted at pH 8.5 for each of the three types of substrates. The pH value of elution corresponds to the approximate isoelectric point of the eluted protein. Immunoabsorption studies with an antibody preparation raised against a purified low pI form confirmed that a 51,000-dalton transferase form, GTM1, eluted primarily at pH 6.7 and that a 54,000-dalton form, GTM2, eluted at pH 8.5. A mathematical treatment of the ratios of activity after 3-methylcholanthrene treatment to that after phenobarbital treatment versus pH produced six patterns of activity. A minimum of two enzymes at the low pH region and one enzyme at the high pH region, all with broad-substrate specificity, could account for these patterns.

Effect of Different Detergent Systems on the Molecular Size of UDP Glucuronosyltransferase and Other Microsomal Drug-Metabolizing Enzymes

Mouse liver microsomes were solubilized in various detergent systems, and the resulting aggregate structures associated with cytochrome P-450, cytochrome c reductase, and UDP glucuronosyltransferase were sized by gel filtration chromatography. Cholate or its derivative, CHAPS, in combination with Emulgen 911 or Lubrol 12A9 were necessary to generate a particle of about 140 k daltons, the smallest structure associated with cytochrome P-450. Cholate or CHAPS alone was sufficient to generate a minimally sized aggregate of 200 k daltons associated with NADPH cytochrome c reductase activity. Cholate in combination with Emulgen 911 or Lubrol 12A9 generated particles of about 280 k daltons associated with UDP glucuronosyltransferase activity. CHAPS alone also generated similarly sized particles under conditions in which UDP glucuronosyltransferase activity toward 1-naphthol and morphine was two to about twenty times greater, respectively, than with the combination of detergents. This finding suggests that the zwitterionic CHAPS is superior to other detergent systems for studies concerned with the purification of transferase enzymes, a microsomal system in which investigation of the number of different forms has been hampered by the instability of the enzyme upon solubilization and subsequent manipulation.

Differential induction of UDP glucuronosyltransferase activities towards various substrates after polycyclic aromatic hydrocarbon administration to rats

Administration of the carcinogens, benzo[a]pyrene and 1,2-benzanthracene, increased UDP glucuronosyltransferase activities towards 4-nitrophenol, 3-hydroxybenzo[a]pyrene, 7-hydroxycoumarin and 1-naphthol to a greater extent than did pretreatment with the noncarcinogens, anthracene and phenanthrene. However, the activity towards morphine was preferentially increased by the noncarcinogens. Activities towards testosterone and oestrone were only slightly increased by the four hydrocarbons. The results suggest that even within a single class of inducer various compounds are capable of causing a differential stimulation of UDP glucuronosyltransferase activities towards certain substrates.

A comparison of the isoelectric points of mouse liver udp- -glucuronosyltransferase enzymes conjugating the twelve benzo[a]pyrene phenols.

Abstract Solubilized mouse liver microsomes were subjected to chromatofocusing using a pH 9.5 to 6.0 gradient. UDP-glucuronosyltransferase activity was assayed using 12 benzo[a]pyrene phenols as substrates. The rank of microsomal activity for the phenols was as follows: 12 > 10 > 4 > 1 > 7 > 5 > 8 > 9 > 3 > 11 > 6 > 2. Fractions separated on chromatofocusing according to isoelectric point indicated that 3-, 10-, 11-, and 12-hydroxybenzo[a]pyrene were conjugated primarily by a high pI (∼8.5) activity(s), 2-, 6-, 8-, and 9-hydroxybenzo[a]pyrene were conjugated primarily by a low pI (∼6.7) activity(s), and 1-, 4-, 5-, and 7-hydroxybenzo[a]pyrene were conjugated equally well by high and low pI forms.

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.