Interaction of uridine 5'-diphosphoglucuronic acid with microsomal UDP-glucuronosyltransferase in primate liver: the facilitating role of uridine 5'-diphospho-N-acetylglucosamine.
Cytosolic uridine 5'-diphosphoglucuronic acid is the essential cosubstrate for all hepatic microsomal UDP-glucuronosyltransferase-mediated reactions. Uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) has been implicated as an activator of UDP-glucuronosyltransferases in vivo, acting either as an allosteric effector or by enhancing access of uridine 5'-diphosphoglucuronic acid to the enzyme. To delineate the interaction of uridine 5'-diphosphoglucuronic acid with microsomal UDP-glucuronosyltransferase and the facilitating role of UDP-GlcNAc, we analyzed bilirubin UDP-glucuronosyltransferase kinetics in microsomes prepared from monkey liver (Macaca fascicularis). Initial rates of bilirubin glucuronide formation were determined by radiochemical assay over a range of uridine 5'-diphosphoglucuronic acid concentrations (0-60 mM), in native microsomes with or without UDP-GlcNAc, or in detergent (digitonin)-pretreated membranes with UDP-GlcNAc. For native microsomes in the absence of UDP-GlcNAc, fitting the data to each of two mathematical models yielded behavior consistent with a single-site model (Km 2.8 mM). In contrast, in the presence of a physiologic concentration (1 mM) of UDP-GlcNAc, analysis of the data excluded the single-site model and was indicative of a non-interactive, two-site (or process) model, characterized by a high-affinity site (Km 0.14 mM) in addition to the low-affinity site. Following detergent-treatment of microsomal membranes, the data were again most consistent with a single low-affinity site.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D014453:Glucuronosyltransferase / Q000378:metabolism*; D007700:Kinetics; D008252:Macaca fascicularis; D008862:Microsomes, Liver / Q000201:enzymology*; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism*; D014537:Uridine Diphosphate N-Acetylglucosamine / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000378:metabolism*
D014539:Uridine Diphosphate Sugars; D014535:Uridine Diphosphate Glucuronic Acid; D014537:Uridine Diphosphate N-Acetylglucosamine; D014453:Glucuronosyltransferase