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

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 (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 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.

Oral tolerization to adenoviral antigens permits long-term gene expression using recombinant adenoviral vectors.

“…In both the human disease and the mutant rats, BUGT 1 deficiency is inherited as an autosomal recessive characteristic and results in life-long unconjugated hyperbilirubinemia leading to cerebral toxicity (17)(18)(19)(20). The advantage of using Gunn rats for developing gene therapy methods is that the expression of BUGT 1 can be conveniently evaluated by monitoring serum bilirubin levels (15). In addition, excretion of bilirubin glucuronides in the bile provides unequivocal evidence of BUGT1 expression in vivo.…”

“…Gunn rats are an animal model of Crigler-Najjar syndrome type I (15,16). In both the human disease and the mutant rats, BUGT 1 deficiency is inherited as an autosomal recessive characteristic and results in life-long unconjugated hyperbilirubinemia leading to cerebral toxicity (17)(18)(19)(20).…”

Induction of central tolerance by intrathymic inoculation of adenoviral antigens into the host thymus permits long-term gene therapy in Gunn rats.

“…Gunn rats lack hepatic uridine-diphospho-glucuronate glucuronosyltransferase (UGT) activity toward bilirubin and, as a consequence, do not excrete conjugated bilirubin in the bile (18). Gunn rats are an animal model of human CriglerNajjar syndrome type I.…”

Immunochemical analysis of uridine diphosphate-glucuronosyltransferase in four patients with the Crigler-Najjar syndrome type I.

“…Microsomes from human kidney are active towards phenolic compounds (27) A possible explanation for the observations in patients A and B is that bilirubin and phenolic compounds are conjugated by one isoenzyme, which shows microheterogeneity on SDS-PAGE and which is absent or mutated in these two patients. However, a number ofconsiderations argue against this explanation: for rat liver it has been shown that the purified bilirubin-conjugating isoenzyme has little or no activity towards PNP and vice versa (13). Furthermore, glucuronidation of bilirubin takes place at carboxyl groups, whereas phenolic compounds like PNP are glucuronidated at aromatic hydroxyl groups.…”

“…The differential induction and development profiles of substrate-specific UDPGT activities are indirect indications for the existence of multiple isoenzymes of UDPGT in the rat (9-1 1). Chromatofocusing of rat liver microsomal UDPGT activities revealed the existence of at least five different isoenzymes (12)(13)(14), and recently two more isoenzymes have been purified (15,16). Two of these isoenzymes are a bilirubin-and phenol-UDPGT.…”