Detection of human pancreatic pro-phospholipase A2 activation using an immunoassay for the free activation peptide DSGISPR.
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.
Gudgeon
A M
AM
Department of Surgery, St George's Hospital Medical School, London, UK.
Patel
G
G
Hermon-Taylor
J
J
Hurley
P
P
Bowyer
R C
RC
Jehanli
A M
AM
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
D009842:Oligopeptides; D010446:Peptide Fragments; D011506:Proteins; C055324:phospholipase A2-activating protein; D010741:Phospholipases A; D054467:Phospholipases A2