Substrate specificity and characterization of rat liver p-nitrophenol, 3 α-hydroxysteroid and 17 β-hydroxysteroid UDP-glucuronosyltransferases

Abstract

Purified preparations of rat liver 17-hydroxysteroid, 3-hydroxyandrogen and p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferases were further characterized as to their substrate specificities, phospholipid-dependency and physical properties. The two steroid UDP-glucuronosyltransferases were shown to exhibit strict stereospecificity with respect to the conjugation of steroids and bile acids. These enzymes have been renamed 17 beta-hydroxysteroid and 3 alpha-hydroxysteroid UDP-glucuronosyltransferase to reflect this specificity for important endogenous substrates. An endogenous substrate has not yet been identified for the p-nitrophenol (3-methylcholanthrene-inducible) UDP-glucuronosyltransferase. The steroid UDP-glucuronosyltransferase activities were dependent on phospholipid for maximal catalytic activity. Complete delipidation rendered the UDP-glucuronosyltransferases inactive, and enzymic activity was not restored when phospholipid was added to the reaction mixture. After partial delipidation, phosphatidylcholine was the most efficient phospholipid for restoration of enzymic activity. Partial delipidation also altered the kinetic parameters of the 3 alpha-hydroxysteroid UDP-glucuronosyltransferase. The three purified UDP-glucuronosyltransferases are separate and distinct proteins, with different amino acid compositions and peptide maps generated by limited proteolysis with Staphylococcus aureus V8 proteinase. Some similarity was observed between the amino acid composition and limited proteolytic maps of the steroid UDP-glucuronosyltransferases, suggesting they are more closely related to each other than to the p-nitrophenol UDP-glucuronosyltransferase.

Purification and characterization of human liver dehydroepiandrosterone sulphotransferase

“…DHEA was the most rapidly sulphated steroid; however, all of the steroids tested, including testosterone and oestrone, were conjugated to some degree. This relative lack of specificity for steroid sulphation differs from the high degree of selectivity observed with the different forms of liver UDP-glucuronyltransferase, where the individual enzymes are very specific for the location and orientation of steroid hydroxy groups which are conjugated (Falany & Tephly, 1983;Falany et al, 1986).…”

Expression of a human liver cDNA encoding a UDP‐glucuronosyltransferase catalysing the glucuronidation of hyodeoxycholic acid in cell culture

“…The 3tz-hydroxy position represents the primary site of conjugation of the steroids lacking 6a-hydroxyl group. Androsterone, which has been shown to be glucuronidated by a specific UDPGT isoenzyme conjugating 3a-steroids in rat and human [18,19], was not apparently a substrate for the expressed HLUG25. It has been shown that monohydroxylated bile acids could also form carboxyl-linked glucuronides [20].…”

Regulation of the Rat UGT1A6 by Glucocorticoids Involves a Cryptic Glucocorticoid Response Element

“…The second cluster containing exons B1 through B5 encodes enzymes with substrate specificities consistent with being members of the neonatal cluster. UGT1A6 is the cluster A member (A1) that encodes an enzyme characterized by having relatively high specific activity with p-nitrophenol as substrate (Falany et al, 1986).…”