Isolation and characterization of hyodeoxycholic‐acid: UDP‐glucuronosyltransferase from human liver

Abstract

The enzyme hyodeoxycholic-acid : UDP-glucuronosyltransferase was purified about 230-fold from a solubilized human liver microsomal preparation utilizing anion-exchange chromatography, ampholyte-displacement chromatography and UDP-hexanolamine -Sepharose affinity chromatography. The homogeneity of the final enzyme preparation was judged by two criteria: the appearance of a single band of M , 52000 in SDS/PAGE; the elution of a single peak in reversed-phase FPLC. The isolated enzyme catalyzed the glucuronidation of the 6a-hydroxy bile acids hyodeoxycholic and hyocholic acids, and of the steroid hormone estriol, with a ratio of relative reaction rates of 13 : 1 : 2.7. UDP-glucuronosyltransferase activities toward the 3a-hydroxy bile acid lithocholic acid, androsterone, testosterone, bilirubin and p-nitrophenol were not detectable in the pure enzyme preparation and were shown to be separated from enzyme activity toward hyodeoxycholic acid during ampholyte-displacement chromatography and/or UDP-hexanolamine -Sepharose affinity chromatography. Two-substrate kinetic analysis of hyodeoxycholic-acid-conjugating activity gave a sequential mechanism with apparent K, values of 12 pM and 4 pM for hyodeoxycholic acid and UDP-glucuronic acid, respectively. Phospholipids were required for reconstitution of maximal activity toward hyodeoxycholic acid. Phosphatidylcholine was the most effective activator of enzyme activity.

Kinetic Characterization of the 1a Subfamily of Recombinant Human Udp-Glucuronosyltransferases

“…The three-dimensional struc-ture of the active site is unknown. A unanimous conclusion of all previous mechanistic studies is that the reaction involves the formation of a ternary complex (Potrepka and Spratt, 1972;Vessey and Zakim, 1972;Sanchez and Tephly, 1975;Rao et al, 1976;Koster and Noordhoek, 1983;Falany et al, 1987;Matern et al, 1991;Yin et al, 1994). Product and dead-end inhibition studies conducted to determine the order of substrate binding have given ambiguous results, however.…”

UDP-Glucuronic Acid Binds First and the Aglycone Substrate Binds Second to Form a Ternary Complex in UGT1A9-Catalyzed Reactions, in Both the Presence and Absence of Bovine Serum Albumin

“…Androsterone and testosterone Rapid equilibrium random-order ternary complex Falany et al, 1987 Purified UGT from human liver Hyodeoxycholic acid Ternary-complex mechanism Matern et al, 1991 and 0.05 to 5 M for entacapone, 4-MU, and 1-naphthol, respectively. After incubation, a 50-l aliquot from each chamber was mixed with 100 l of ice-cold 4 M perchloric acid/methanol (1:5 mix), placed at Ϫ18°C for 30 min, and centrifuged (at room temperature) for 5 min at 16000g, and the test compound concentration in the resulting supernatant was determined by highperformance liquid chromatography (HPLC) analyses.…”

“…To understand the overall enzyme kinetic mechanism, we have also investigated the reverse reaction, an approach that was rarely taken in previous UGT studies, particularly because recombinant UGTs became available and ensure that the analysis is conducted on a single enzyme rather than a mixture of different UGTs (Vessey and Zakim, 1972;Rao et al, 1976;Matern et al, 1991). Hence, the bisubstrate enzyme kinetics, in the presence of BSA, was also studied for the reverse reaction, namely the formation of 4-MU and UDPGA from 4-MUG and UDP (Fig.…”

“…Alongside the broad agreement on the formation of ternary complex, however, there is disagreement among the previous studies on whether the two substrates, the aglycone substrate and the UDPGA, bind in a random order or in a compulsory order (Table 1). The observed discrepancies in the order of substrate binding may have arisen from the use of liver microsomal preparations that contain multiple UGT enzymes (Potrepka and Spratt, 1972;Vessey and Zakim, 1972;Sanchez and Tephly, 1975;Rao et al, 1976;Koster and Noordhoek, 1983) or of partially purified UGT enzymes (Matern et al, 1982(Matern et al, , 1991Falany et al, 1987;Yin et al, 1994) that may have been inactivated by detergents during the purification process (Kurkela et al, 2003).…”

Cytosylglucuronic acid synthase (cytosine: UDP-glucuronosyltransferase) from Streptomyces griseochromogenes, the first prokaryotic UDP-glucuronosyltransferase

“…Neither uracil and adenine nor 4-nitrophenol and ot-naphthol, the two most commonly used substrates for assaying eukaryotic UDP-GTs, could replace cytosine. The eukaryotic and prokaryotic enzymes are all monomers with moderate sizes (2,9,22,27). Like the eukaryotic enzymes, CGA synthase has no metal requirement.…”