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-phosphocholine ([H-3]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 [H-3]acetyl-PAF on the PVDF membranes with an ultrahigh-sensitivity TV camera system. Enzyme activity is detected as a decrease in beta-radioluminescence of [H-3]acetyl-PAF on the blotted membranes, since of [H-3]acetyl-PAF remains on the membrane but the [H-3]acetate released by the enzyme reaction detaches from the membrane. This method has been used to identify PAF acetylhydrolase and phospholipase A(2). (C) 1994 Academic Press, Inc.