TY - JOUR
T1 - Isolation and characterization of multiple forms of rat liver UDP-glucuronate glucuronosyltransferase
AU - Roy Chowdhury, J.
AU - Roy Chowdhury, N.
AU - Falany, C. N.
AU - Tephly, T. R.
AU - Arias, I. M.
PY - 1986
Y1 - 1986
N2 - 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.
AB - 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.
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U2 - 10.1042/bj2330827
DO - 10.1042/bj2330827
M3 - Article
C2 - 3085655
AN - SCOPUS:0022627563
SN - 0264-6021
VL - 233
SP - 827
EP - 837
JO - Biochemical Journal
JF - Biochemical Journal
IS - 3
ER -