Purification and properties of UDP-glucuronyltransferase from kidney microsomes of beta-naphthoflavone-treated rat

J Biochem. 1989 Aug;106(2):248-52. doi: 10.1093/oxfordjournals.jbchem.a122839.

Abstract

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)

MeSH terms

  • Acetylglucosaminidase / metabolism
  • Amino Acid Sequence
  • Animals
  • Benzoflavones / pharmacology*
  • Catalysis
  • Flavonoids / pharmacology*
  • Glucuronosyltransferase / isolation & purification*
  • Glucuronosyltransferase / metabolism
  • Glycoproteins / analysis
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • 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
  • beta-Naphthoflavone

Substances

  • Benzoflavones
  • Flavonoids
  • Glycoproteins
  • Isoenzymes
  • beta-Naphthoflavone
  • Glucuronosyltransferase
  • Acetylglucosaminidase
  • Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase