Abstract
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 polypep-tide 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-glucuronyltrans-ferase. 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 was 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+.
