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-[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.
Akiyama
M
M
Department of Biochemistry, School of Pharmaceutical Science, University of Shizuoka, Japan.
Sugatani
J
J
Suzuki
Y
Y
Miwa
M
M
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D001798:Blood Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006868:Hydrolysis; D015151:Immunoblotting; D008163:Luminescent Measurements; D008567:Membranes, Artificial; D008722:Methods; D010741:Phospholipases A / Q000097:blood / Q000378:metabolism*; D054467:Phospholipases A2; D011145:Polyvinyls; D011827:Radiation; D012680:Sensitivity and Specificity; D012967:Sodium Dodecyl Sulfate; D013690:Television / Q000295:instrumentation*; D014316:Tritium; D005465:Fluorocarbon Polymers
D043203:1-Alkyl-2-acetylglycerophosphocholine Esterase; D001798:Blood Proteins; D008567:Membranes, Artificial; D010741:Phospholipases A; D054467:Phospholipases A2; D011145:Polyvinyls; D012967:Sodium Dodecyl Sulfate; D014316:Tritium; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers