Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase

Jayanta Roy-Chowdhury, N. Roy Chowdhury, C. N. Falany, T. R. Tephly, I. M. Arias

Research output: Contribution to journalArticle

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Abstract

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (M(r) 51,000), Ib (M(r) 52,000), Ic (M(r) 56,000), II (M(r) 52,000), IV (M(r) 53,000) and V (M(r) 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of M(r) 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct 'maps', indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.

Original languageEnglish (US)
Pages (from-to)827-837
Number of pages11
JournalBiochemical Journal
Volume233
Issue number3
StatePublished - 1986

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Glucuronosyltransferase
Glucuronic Acid
Uridine Diphosphate
Liver
Rats
Protein Isoforms
Transferases
Sepharose
Nonoxynol
Proteolysis
Affinity chromatography
Agarose Chromatography
Peptides
Periodic Acid
Substrates
Substrate Specificity
Chromatography
Electrophoresis
Phosphatidylcholines
Affinity Chromatography

ASJC Scopus subject areas

  • Biochemistry

Cite this

Roy-Chowdhury, J., Roy Chowdhury, N., Falany, C. N., Tephly, T. R., & Arias, I. M. (1986). Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase. Biochemical Journal, 233(3), 827-837.

Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase. / Roy-Chowdhury, Jayanta; Roy Chowdhury, N.; Falany, C. N.; Tephly, T. R.; Arias, I. M.

In: Biochemical Journal, Vol. 233, No. 3, 1986, p. 827-837.

Research output: Contribution to journalArticle

Roy-Chowdhury, J, Roy Chowdhury, N, Falany, CN, Tephly, TR & Arias, IM 1986, 'Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase', Biochemical Journal, vol. 233, no. 3, pp. 827-837.
Roy-Chowdhury, Jayanta ; Roy Chowdhury, N. ; Falany, C. N. ; Tephly, T. R. ; Arias, I. M. / Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase. In: Biochemical Journal. 1986 ; Vol. 233, No. 3. pp. 827-837.
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