Smit N P, Pavel S, Kammeyer A, Westerhof W
Department of Dermatology, University of Amsterdam, The Netherlands.
Anal Biochem. 1990 Nov 1;190(2):286-91. doi: 10.1016/0003-2697(90)90195-f.
A new sensitive method for the determination of catechol O-methyltransferase activity has been developed. The method is based on the O-methylation of the indolic intermediates of melanin metabolism. The substrate, 5,6-dihydroxyindole-2-carboxylic acid, is converted by the enzyme to two O-methylated products, which can be separated by high-performance liquid chromatography and measured with fluorimetric detection. The physiological presence of both substrate and products could be detected in crude melanoma cell extracts. The limit of sensitivity for detection of the O-methylated products is less than 0.5 pmol per injection. The method was compared with an earlier described HPLC method which makes use of uv detection of O-methylated products of 3,4-dihydroxybenzoic acid. The described method will be used to study the importance of catechol O-methyltransferase as a protective enzyme in (malignant) melanocytes.
已开发出一种用于测定儿茶酚-O-甲基转移酶活性的新的灵敏方法。该方法基于黑色素代谢中吲哚中间体的O-甲基化。底物5,6-二羟基吲哚-2-羧酸被该酶转化为两种O-甲基化产物，它们可通过高效液相色谱分离并用荧光检测法进行测定。在黑色素瘤细胞粗提物中可检测到底物和产物的生理存在。每次进样检测O-甲基化产物的灵敏度极限小于0.5皮摩尔。将该方法与先前描述的利用紫外检测3,4-二羟基苯甲酸O-甲基化产物的高效液相色谱法进行了比较。所描述的方法将用于研究儿茶酚-O-甲基转移酶作为（恶性）黑素细胞中一种保护酶的重要性。
