Immunochemical detection of 'platelet type' phospholipase A2 in the rat.
Polyclonal antibodies were raised against rat platelet phospholipase A2. One of them, designated as R377 was prepared by immunizing a rabbit with the intact enzyme. The other antibody, designated as R385, was prepared by immunizing with the enzyme treated with 2-mercaptoethanol. The antibody R377 bound to rat platelet phospholipase A2 almost exclusively, while the antibody R385 reacted not only with rat platelet phospholipase A2 but with the enzymes obtained from snake venom or mammalian pancreas. The antibody R377 bound to the non-reduced rat platelet phospholipase A2 bearing intact intramolecular disulfide bonds, but not to the reduced enzyme. In contrast, the antibody R385 reacted with both non-reduced and reduced enzymes. R377 may recognize the conformational structure of the enzyme. Both antibodies inhibited the enzyme activity. The antibody R377, but not R385, interfered with the interaction of the enzyme with heparin, which is one of the characteristic properties of rat platelet phospholipase A2. The antibody R377 reacted with phospholipases A2 of bone-marrow cells and of peritoneal exudated cells prepared from caseinate-treated rats, indicating that some myeloid cells other than platelets also contain 'platelet type' phospholipase A2. An immunochemical method for measurement of rat 'platelet type' phospholipase A2 was developed. The sensitivity of this method was 10 ng/ml of phospholipase A2 in the preparation. One of the advantages of the present immunochemical method is that the measurement was not affected by the presence of an endogenous inhibitor(s) of enzymatic activity.
Murakami
M
M
Department of Health Chemistry, Faculty of Pharmaceutical Sciences, University of Tokyo, Japan.
Kudo
I
I
Natori
Y
Y
Inoue
K
K
D000818:Animals; D000918:Antibody Specificity; D001202:Ascitic Fluid / Q000201:enzymology; D001792:Blood Platelets / Q000201:enzymology*; D001853:Bone Marrow / Q000201:enzymology; D004797:Enzyme-Linked Immunosorbent Assay; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007150:Immunohistochemistry; D008297:Male; D008533:Megakaryocytes / Q000201:enzymology; D008623:Mercaptoethanol / Q000494:pharmacology; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D010740:Phospholipases / Q000097:blood*; D010741:Phospholipases A / Q000032:analysis / Q000097:blood* / Q000276:immunology; D054467:Phospholipases A2; D011489:Protein Denaturation; D051381:Rats; D011919:Rats, Inbred Strains; D014018:Tissue Distribution
D007106:Immune Sera; D008623:Mercaptoethanol; D010740:Phospholipases; D010741:Phospholipases A; D054467:Phospholipases A2