title: Detection of human pancreatic pro-phospholipase A2 activation using an immunoassay for the free activation peptide DSGISPR.
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 chromatographed 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.
authors.affiliation: Department of Surgery, St George's Hospital Medical School, London, UK.
authors.forename: A M
authors.initials: AM
authors.lastname: Gudgeon
authors.forename: G
authors.initials: G
authors.lastname: Patel
authors.forename: J
authors.initials: J
authors.lastname: Hermon-Taylor
authors.forename: P
authors.initials: P
authors.lastname: Hurley
authors.forename: R C
authors.initials: RC
authors.lastname: Bowyer
authors.forename: A M
authors.initials: AM
authors.lastname: Jehanli
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D002846:Chromatography, Affinity; D004789:Enzyme Activation; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000032:analysis*; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000032:analysis; D010741:Phospholipases A / Q000032:analysis*; D054467:Phospholipases A2; D011506:Proteins / Q000032:analysis*; D011817:Rabbits; D011863:Radioimmunoassay
chemical_list: D009842:Oligopeptides; D010446:Peptide Fragments; D011506:Proteins; C055324:phospholipase A2-activating protein; D010741:Phospholipases A; D054467:Phospholipases A2