Determination of platelet-activating factor acetylhydrolase activity by blotting, beta-radioluminescence, and ultrahigh-sensitivity television camera detection

Anal Biochem. 1994 May 1;218(2):295-9. doi: 10.1006/abio.1994.1181.

Abstract

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.

MeSH terms

  • 1-Alkyl-2-acetylglycerophosphocholine Esterase
  • Blood Proteins / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Hydrolysis
  • Immunoblotting*
  • Luminescent Measurements*
  • Membranes, Artificial
  • Methods
  • Phospholipases A / blood
  • Phospholipases A / metabolism*
  • Phospholipases A2
  • Polyvinyls
  • Radiation
  • Sensitivity and Specificity
  • Sodium Dodecyl Sulfate
  • Television / instrumentation*
  • Tritium

Substances

  • Blood Proteins
  • Membranes, Artificial
  • Polyvinyls
  • Tritium
  • polyvinylidene fluoride
  • Sodium Dodecyl Sulfate
  • Phospholipases A
  • Phospholipases A2
  • 1-Alkyl-2-acetylglycerophosphocholine Esterase