使用带荧光检测的高效液相色谱法进行羟基吲哚-O-甲基转移酶活性测定：测定由N-乙酰血清素和S-腺苷-L-甲硫氨酸酶促形成的褪黑素。

Hydroxyindole-O-methyltransferase activity assay using high-performance liquid chromatography with fluorometric detection: determination of melatonin enzymatically formed from N-acetylserotonin and S-adenosyl-L-methionine.

作者信息

Itoh M T, Hattori A, Sumi Y

机构信息

Department of Chemistry, St. Marianna University School of Medicine, Miyamae-ku, Kawasaki, Japan.

出版信息

J Chromatogr B Biomed Sci Appl. 1997 Apr 25;692(1):217-21. doi: 10.1016/s0378-4347(96)00503-8.

DOI:10.1016/s0378-4347(96)00503-8

PMID:9187403

Abstract

A reliable, sensitive and rapid assay has been developed for determining the activity of hydroxyindole-O-methyltransferase (HIOMT; S-adenosyl-L-methionine:N-acetylserotonin-O-methyltransferase; EC 2.1.1.4), which catalyzes the final step in the melatonin (N-acetyl-5-methoxytryptamine) biosynthetic pathway. This method is based on the separation and detection of melatonin formed enzymatically from N-acetylserotonin and S-adenosyl-L-methionine, by high-performance liquid chromatography with fluorometric detection. The detection limit for melatonin formed per sample was as low as 150 fmol, indicating that the sensitivity of this assay was comparable to that of a radioisotopic assay. The assay was applied to the determination of HIOMT activity in rat pineal gland. The HIOMT activity obtained in this study was comparable with, or slightly lower than those reported previously using radioisotopic assays.

摘要

已开发出一种可靠、灵敏且快速的检测方法，用于测定羟基吲哚 - O - 甲基转移酶（HIOMT；S - 腺苷 - L - 甲硫氨酸：N - 乙酰血清素 - O - 甲基转移酶；EC 2.1.1.4）的活性，该酶催化褪黑素（N - 乙酰 - 5 - 甲氧基色胺）生物合成途径的最后一步。此方法基于通过高效液相色谱荧光检测法，对由N - 乙酰血清素和S - 腺苷 - L - 甲硫氨酸酶促形成的褪黑素进行分离和检测。每个样品形成的褪黑素的检测限低至150飞摩尔，表明该检测方法的灵敏度与放射性同位素检测方法相当。该检测方法应用于大鼠松果体中HIOMT活性的测定。本研究中获得的HIOMT活性与先前使用放射性同位素检测方法报道的结果相当，或略低。