Type 1-prophospholipase A2 propeptide immunoreactivity is released from activated granulocytes.
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.


CONCLUSIONS
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.
Rae
D
D
Department of Surgery, St. George's Hospital Medical School, London, UK.
Sumar
N
N
Beechey-Newman
N
N
Gudgeon
M
M
Hermon-Taylor
J
J
D000595:Amino Acid Sequence; D016207:Cytokines / Q000378:metabolism; D004792:Enzyme Precursors; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006098:Granulocytes / Q000737:chemistry / Q000201:enzymology* / Q000502:physiology; D006801:Humans; D008214:Lymphocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D008969:Molecular Sequence Data; D009000:Monocytes / Q000737:chemistry / Q000201:enzymology / Q000378:metabolism; D010741:Phospholipases A / Q000097:blood / Q000276:immunology* / Q000493:pharmacokinetics*; D054467:Phospholipases A2; D011498:Protein Precursors / Q000097:blood / Q000276:immunology* / Q000493:pharmacokinetics*; D014357:Trypsin / Q000737:chemistry
D016207:Cytokines; D004792:Enzyme Precursors; D011498:Protein Precursors; D010741:Phospholipases A; D054467:Phospholipases A2; C085333:prophospholipase A2; D014357:Trypsin