Determination of Platelet-Activating Factor Acetylhydrolase Activity by Blotting, β-Radioluminescence, and Ultrahigh-Sensitivity Television Camera Detection
✍ Scribed by M. Akiyama; J. Sugatani; Y. Suzuki; M. Miwa
- Publisher
- Elsevier Science
- Year
- 1994
- Tongue
- English
- Weight
- 469 KB
- Volume
- 218
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
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.