Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat.
Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4-5-fold by treatment of the animal with beta-naphthoflavone. The transferase activity toward serotonin, an endogenous substrate, was also enhanced about 7.5-fold. A form of UDP-glucuronyltransferase was purified from kidney microsomes of beta-naphthoflavone-treated rat by solubilization with sodium cholate and two steps of column chromatography, the first with DEAE-Toyopearl (fast flow rate liquid chromatography:FFLC) and the second with UDP-hexanolamine Sepharose 4B (affinity chromatography). These procedures gave about 39-fold purification and 11.5% yield of the transferase activity toward 1-naphthol. The preparation, tentatively termed "GT-2," was highly purified as judged from the single protein band (Mr 54,000) on sodium dodecylsulfate (SDS)-polyacrylamide slab gel electrophoresis. It catalyzed the glucuronidation of not only phenolic xenobiotics such as 1-naphthol, 4-nitrophenol, and 4-methylumbelliferone but also serotonin. From the result that apparent molecular weight of GT-2 was reduced to 50,000 by endo-beta-N-acetylglucosaminidase H (Endo H)-treatment, GT-2 was found to be a 50,000 Da polypeptide carrying "high mannose" type oligosaccharide chain(s). The NH2-terminal sequence of 20 residues of GT-2 was determined to be Asp-Lys-Leu-Leu-Val-Val-Pro-Gln-Asp-Gly-Ser-His-Trp-Leu-Ser-Met-Lys-Glu- Ile-Val . It was observed that there are two amino acids substitutions in the seven NH2-terminal residues in comparison with GT-1, which was purified from liver microsomes of 3-methylcholanthrene-treated rat. The NH2-terminal sequence of GT-2 was found to be homologous with the NH2-terminal sequence from the 26th to 46th amino acid residue of various UDP-glucuronyltransferase cloned by other investigators.(ABSTRACT TRUNCATED AT 250 WORDS)
Yokota
H
H
Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido.
Ohgiya
N
N
Ishihara
G
G
Ohta
K
K
Yuasa
A
A
D000118:Acetylglucosaminidase / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001571:Benzoflavones / Q000494:pharmacology*; D002384:Catalysis; D005419:Flavonoids / Q000494:pharmacology*; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008861:Microsomes / Q000187:drug effects / Q000201:enzymology*; D008862:Microsomes, Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D051381:Rats; D011919:Rats, Inbred Strains; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D005419:Flavonoids; D006023:Glycoproteins; D007527:Isoenzymes; D019324:beta-Naphthoflavone; D014453:Glucuronosyltransferase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase