Purification and properties of 5-hydroxytryptamine UDP-glucuronyltransferase from rat liver microsomes.
5-Hydroxytryptamine UDP-glucuronyltransferase was highly purified from untreated rat liver microsomes. The specific activity towards 5-hydroxytryptamine was increased 178-fold over the starting solubilized microsomes with a final yield of 3%. The final preparation contained two major and one minor Coomassie brilliant blue staining polypeptide bands visible after SDS-polyacrylamide gel electrophoresis. One of the major bands was identified as 3-methylcholanthrene-inducible UDP-glucuronyltransferase, so the other (molecular weight of 55,500) appeared to be 5-hydroxytryptamine UDP-glucuronyltransferase. Concanavalin A reacted with the 55,500-dalton polypeptide. Phospholipid was indispensable for the enzyme activity. The enzyme activity in the final preparation was activated by divalent cations. Simple Michaelis-Menten kinetics were followed with respect to 5-hydroxytryptamine, but deviations from this kinetics were observed with respect to UDP-glucuronic acid and Mg2+. As regards Mg2+ stimulation, further experiments indicated that the added Mg2+ was non-competitive with 5-hydroxytryptamine, but at low concentrations of Mg2+ it was competitive with UDP-glucuronic acid and at high concentrations of Mg2+ it was non-competitive with UDP-glucuronic acid. The final preparation showed high substrate specificity towards 5-hydroxytryptamine among endogenous substrates tested. From these results, it was concluded that the enzyme described here is a new form of UDP-glucuronyltransferase isozyme, and its activity showed a peculiar dependence on Mg2+.
Abe
N
N
Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido.
Abe
E
E
Yuasa
A
A
D000818:Animals; D002413:Cations, Divalent; D004591:Electrophoresis, Polyacrylamide Gel; D014453:Glucuronosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D006023:Glycoproteins / Q000032:analysis; D008081:Liposomes; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D051381:Rats; D011919:Rats, Inbred Strains; D012701:Serotonin / Q000378:metabolism*; D013379:Substrate Specificity; D014535:Uridine Diphosphate Glucuronic Acid / Q000378:metabolism
D002413:Cations, Divalent; D006023:Glycoproteins; D008081:Liposomes; D014535:Uridine Diphosphate Glucuronic Acid; D012701:Serotonin; D014453:Glucuronosyltransferase