An improved assay method for UDP-glucuronyltransferase activity towards 5-hydroxytryptamine and the characteristic properties of the enzyme.
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 Mg(2+)-UDPGA complex which showed higher affinity for the enzyme than UDPGA at the low concentration of Mg2+ and further stimulated by the formation of Mg(2+)-enzyme complex at the higher concentration of Mg2+.
Abe
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Department of Veterinary Biochemistry, School of Veterinary Medicine, Rakuno Gakuen University, Hokkaido, Japan.
Abe
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Yuasa
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A
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D004492:Edetic Acid / Q000494:pharmacology; D014453:Glucuronosyltransferase / Q000032:analysis* / Q000187:drug effects / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D015203:Reproducibility of Results; D012701:Serotonin / Q000378:metabolism*
D012701:Serotonin; D004492:Edetic Acid; D014453:Glucuronosyltransferase; D008274:Magnesium; D002118:Calcium