EXPRESSION OF UDP-GLUCURONOSYLTRANSFERASE CDNA IN SACCHAROMYCES-CEREVISIAE AS A MEMBRANE-BOUND AND AS A CYTOSOLIC FORM

被引:18
作者
TOGHROL, F
KIMURA, T
OWENS, IS
机构
[1] NICHHD,OFF SCI DIRECTOR,DRUG BIOTRANSFORMAT SECT,BETHESDA,MD 20892
[2] NATL CANC CTR,RES INST,DIV BIOPHYS,CHUO KU,TOKYO 104,JAPAN
关键词
D O I
10.1021/bi00461a020
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
The mouse clone UDPGTm-1 encodes a UDP-glucuronosyltransferase enzyme which was isolated from a λgt11 cDNA library constructed with phenobarbital-induced liver mRNA [Kimura, T., & Owens, I. S. (1987) Eur. J. Biochem. 168, 515-521]. In order to establish substrate specificity, UDPGTm-1 was inserted into the yeast vector pEVP11 and expressed in Saccharomyces cerevisiae strain AH22. Cells transformed with the expression unit pUDPGTm-1c (insert in correct orientation with respect to promoter) stably transcribe the transferase cDNA. Consistent with the presence of mRNA, pUDPGTm-1c-transformed AH22 cells synthesize a transferase protein with Mr ≃ 51000 by Western immunoblot analysis. The membrane-bound transferase expressed in yeast in glycosylated as indicated by its enhanced electrophoretic mobility in a SDS-polyacrylamide gel following endoglycosidase H treatment and detection by Western immunoblot analysis. A survey, using 12 aglycons in an assay with microsomes from cells which express the protein, shows preferential glucuronidation of naphthol and estrone followed by p-nitrophenol. Testosterone, phenolphthalein, dihydrotestosterone, androsterone, and 4-methylumbelliferone are conjugated at an intermediate level. There is barely detectable glucuronidation of 3-hydroxy- and 9-hydroxybenzo- [a]pyrene and no detectable conversion of morphine or lithocholic acid. The truncated cDNA (lacking the putative membrane-insertion signal-peptide coding sequence, but with a newly adapted translation-start codon) is ligated into pAAH5 and is expressed as a cytosolic transferase form in the protease-deficient ZA521 strain of S. cerevisiae. The Mr ≃ 51000-52000 is similar to that seen in microsomes from AH22 cells where the protein is presumably processed as it is inserted into the membrane. This cytosolic form expresses similar catalytic activity toward naphthol and 3-hydroxybenzo[a]pyrene, very much lower activity toward estrone, p-nitrophenol, and phenolphthalein, and detectable activity toward lithocholic acid when compared to the membrane-bound transferase in AH22 cells. A comparison of the catalytic activity as the membrane-bound form when expressed in AH22 versus ZA521 shows that the relative substrate profile is similar, but higher levels of the transferase protein and activities are seen in AH22 than in ZA521. The transferase as a membrane-bound form glucuronidates at least three substrates traditionally classified as type I, one classified as type II, and at least four steroid aglycons. In contrast, the enzyme, when distributed into the yeast cytosol, has a more restricted substrate range and catalyzes most of these substrates at a much lower rate. © 1990, American Chemical Society. All rights reserved.
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页码:2349 / 2356
页数:8
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