Purification and Properties of UDP-Glucuronyltransferase from Kidney Microsomes of β-Naphthoflavone-Treated Rat

• Published in: Journal of biochemistry (Tokyo) Vol. 106; no. 2; pp. 248 - 252
• Main Authors: Yokota, Hiroshi, Ohgiya, Nobuyuki, Ishihara, Goshi, Ohta, Kazuhide, Yuasa, Akira
• Format: Journal Article
• Language: English
• Published: Oxford Oxford University Press 01.08.1989
• Subjects: Acetylglucosaminidase - metabolism; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; Benzoflavones - pharmacology; beta -naphthoflavone; Biological and medical sciences; Catalysis; Enzymes and enzyme inhibitors; Flavonoids - pharmacology; Fundamental and applied biological sciences. Psychology; Glucuronosyltransferase - isolation & purification; Glucuronosyltransferase - metabolism; Glycoproteins - analysis; Isoenzymes - isolation & purification; Isoenzymes - metabolism; kidney; Kidney - drug effects; Kidney - enzymology; Male; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Microsomes - drug effects; Microsomes - enzymology; Microsomes, Liver - enzymology; Molecular Sequence Data; Rats; Rats, Inbred Strains; Transferases; Purification; Rat; Gel electrophoresis; Enzyme; Rodentia; UDPglucuronosyltransferase; Activation; Microsome; Chromatography; Kidney; Vertebrata; Mammalia; Enzymatic activity; Substrate specificity
• ISSN: 0021-924X; 1756-2651
• DOI: 10.1093/oxfordjournals.jbchem.a122839

Rat kidney microsomal UDP-glucuronyltransferase activities toward phenoic xenobiotics were enhanced about 4–5-fold by treatment of the animal with β-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 β-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)-polyacryl-amide 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-β-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. The notion that the NH2-terminal region to the 25th amino acid resiue of newly synthesized transferase contains the signal peptide was thus confirmed by this study.