OBJECTIVE: To establish a ELISA assay to measure release of type 1-phospholipase A2 propeptide from activated granulocytes. Human type 1-prophospholipase A2 (1-proPLA2) is biosynthesized and stored as inactive zymogen. Activation involves tryptic-like cleavage at the N-terminus, with equimolar release of the heptapeptide DSGISPR. METHODS: Using antibodies directed to the carboxyterminus of synthetic DSGISPR we developed a sensitive solid-phase ELISA specific for the released propeptide that accurately reports the activation of 1-proPLA2. The presence of the 1-proPLA2 precursor itself can be determined by trypsinization of the sample and subsequent assay for free DSGISPR. RESULTS: Using this ELISA, we demonstrated the presence of immunoreactive DSGISPR and its 14 kDa 1-proPLA2-like precursor in human granulocytes, but their absence in human macrophages and lymphocytes. Stimulation of cultured granulocytes with 1 pM of TNF alpha or GM-CSF caused rapid release of DSGISPR and precursor into the surrounding medium. The immunoreactive signal coeluted with standard synthetic DSGISPR on G50 Sephadex chromatography. CONCLUSION: Release of DSGISPR immunoreactivity appears to be a specific consequence of granulocyte activation of potential relevance to the clinical pathophysiology of conditions like acute lung injury.
OBJECTIVE: To establish a ELISA assay to measure release of type 1-phospholipase A2 propeptide from activated granulocytes. Human type 1-prophospholipase A2 (1-proPLA2) is biosynthesized and stored as inactive zymogen. Activation involves tryptic-like cleavage at the N-terminus, with equimolar release of the heptapeptide DSGISPR. METHODS: Using antibodies directed to the carboxyterminus of synthetic DSGISPR we developed a sensitive solid-phase ELISA specific for the released propeptide that accurately reports the activation of 1-proPLA2. The presence of the 1-proPLA2 precursor itself can be determined by trypsinization of the sample and subsequent assay for free DSGISPR. RESULTS: Using this ELISA, we demonstrated the presence of immunoreactive DSGISPR and its 14 kDa 1-proPLA2-like precursor in human granulocytes, but their absence in human macrophages and lymphocytes. Stimulation of cultured granulocytes with 1 pM of TNF alpha or GM-CSF caused rapid release of DSGISPR and precursor into the surrounding medium. The immunoreactive signal coeluted with standard synthetic DSGISPR on G50 Sephadex chromatography. CONCLUSION: Release of DSGISPR immunoreactivity appears to be a specific consequence of granulocyte activation of potential relevance to the clinical pathophysiology of conditions like acute lung injury.