DETERMINATION OF PLATELET-ACTIVATING-FACTOR ACETYLHYDROLASE ACTIVITY BY BLOTTING, BETA-RADIOLUMINESCENCE, AND ULTRAHIGH-SENSITIVITY TELEVISION CAMERA DETECTION

A simple method for detecting the 1-alkyl-2-[H-3]acetyl-sn-glycero-3-p hosphocholine ([H-3]acetyl-PAF)-hydrolyzing activity of serum proteins is described. These were separated by sodium dodecyl sulfate-polyacry lamide gel electrophoresis and transferred to polyvinylidene difluorid e (PVDF) membranes. The assay involves measurement of the beta-radiolu minescence of [H-3]acetyl-PAF on the PVDF membranes with an ultrahigh- sensitivity TV camera system. Enzyme activity is detected as a decreas e in beta-radioluminescence of [H-3]acetyl-PAF on the blotted membrane s, since of [H-3]acetyl-PAF remains on the membrane but the [H-3]aceta te released by the enzyme reaction detaches from the membrane. This me thod has been used to identify PAF acetylhydrolase and phospholipase A (2). (C) 1994 Academic Press, Inc.