An Improved Assay Method for UDP-Glucuronyltransferase Activity towards 5-Hydroxytryptamine and the Characteristic Properties of the Enzyme

[摘要] References(17)Cited-By(1)A simple and reproducible assay method for UDP-glucuronyltransferase (GT) towards 5-hydroxytryptamine (5-HT) was developed. It consists of the removal of unconjugated 5-HT by 0.6 N NH4OH-saturated n-amyl alcohol and the colorimetric estimation of 5-HT glucuronide. Using this assay method, some properties of the enzyme activity in rat liver microsomes were studied. Simple Michaelis-Menten kinetics was followed with respect to 5-HT and the apparent Km value for 5-HT was 0.1 mM. However, the deviation from this kinetics was observed with respect to UDP-glucuronic acid (UDPGA). The apparent Km values for UDPGA were 0.6 mM and 5 mM. The enzyme activity was stimulated by divalent cations. For Mg2+, the enzyme did not obey this kinetics, and the apparent Km values for Mg2+ were 1 mM and 10 mM. In the presence of Mg2+, the apparent Km value for 5-HT did not change but the Vmax value increased. On the other hand, the addition of a low concentration of Mg2+ decreased the apparent Km value for UDPGA and increased the Vmax value. The addition of a high concentration of Mg2+ did not change the apparent Km value for UDPGA but increased the Vmax value. These results indicate that the enzyme activity is stimulated by the formation of Mg2+-UDPGA complex which showed higher affinity for the enzyme than UDPGA at the low concentration of Mg2+ and further stimulated by the formation of Mg2+-enzyme complex at the higher concentration of Mg2+.

[学科分类] 兽医学

[关键词] assay method;enzyme kinetics;magnesium ion;UDP-glucuronyltransferase;5-hydroxytryptamine