Abstract
There are several forms of the enzyme phosphoLipase A2 (PLA2) in human tissues. In the pancreas the enzyme is produced as a zymogen, pro-phospholipase A2 (pro-PLA2). The active form is generated upon proteolytic cleavage of the N-terminal prophospholipase A2 activation peptide (PLAP), with the sequence Asp-Ser-Gly-Ile-Ser-Pro-Arg (DSGISPR). Antisera specific for free PLAP were produced by immunization with the synthetic peptide, N-terminally conjugated to bovine thyroglobin. Affinity purified antibodies were used to develop a radioimmunoassay with a detection limit of 5 nmol/L. Competitive inhibition studies with amino-terminally truncated sequences showed that, at least, the C-terminal pentapeptide (GISPR) was required for significant inhibition. Anti-PLAP antibodies did not react with native human pancreatic homogenate (a source of pro-PLA2). A large immunoreactive signal was generated upon trypsinization, which coeluted with synthetic PLAP when cromatographed on Sephadex-G25. Likewise, Sephadex-G50 chromatograph fractions of the untrypsinized homogenate reacted with the antibodies only after trypsinization. The immunoreactive signal appeared at a molecular weight of 14 500 which corresponds to the reported molecular weight of pancreatic pro-PLA2. This demonstrates that the assay is specific for the free peptide and reports pro-PLA2 activation. PLAP assay may therefore contribute to the study of the role of the PLA2 activation event in disease states such as pancreatitis.