Akiyama M, Sugatani J, Suzuki Y, Miwa M
Department of Biochemistry, School of Pharmaceutical Science, University of Shizuoka, Japan.
Anal Biochem. 1994 May 1;218(2):295-9. doi: 10.1006/abio.1994.1181.
A simple method for detecting the 1-alkyl-2-[3H]-acetyl-sn-glycero-3-phosphocholine ([3H]acetyl-PAF)-hydrolyzing activity of serum proteins is described. These were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes. The assay involves measurement of the beta-radioluminescence of [3H]acetyl-PAF on the PVDF membranes with an ultra-high-sensitivity TV camera system. Enzyme activity is detected as a decrease in beta-radioluminescence of [3H]-acetyl-PAF on the blotted membranes, since [3H]acetyl-PAF remains on the membrane but the [3H]acetate released by the enzyme reaction detaches from the membrane. This method has been used to identify PAF acetylhydrolase and phospholipase A2.
本文描述了一种检测血清蛋白1-烷基-2-[³H]-乙酰基-sn-甘油-3-磷酸胆碱（[³H]乙酰基-PAF）水解活性的简单方法。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离这些蛋白，并转移至聚偏二氟乙烯（PVDF）膜上。该测定方法涉及使用超高灵敏度电视摄像系统测量PVDF膜上[³H]乙酰基-PAF的β放射性发光。酶活性通过印迹膜上[³H]乙酰基-PAF的β放射性发光降低来检测，因为[³H]乙酰基-PAF保留在膜上，但酶反应释放的[³H]乙酸从膜上脱离。该方法已用于鉴定PAF乙酰水解酶和磷脂酶A2。
