Isolation of a protein labeled with diisopropyl fluorophosphate on stimulation of polymorphonuclear leukocytes with immune complexes.
As demonstrated by others, diisopropyl fluorophosphate (DFP) markedly inhibits the O2- generation from guinea-pig polymorphonuclear leukocytes (PMN) stimulated by an antibody complex with ovalbumin (Ag-Ab complex), and also the intracellular uptake of antibody-sensitized erythrocytes by the cells. However, when PMN were treated with DFP and washed to remove the inhibitor, they again became able to exhibit the O2- -generating and phagocytic activities. The [3H]DFP-labeling of intact PMN followed by solubilization with Triton N101, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the existence of several [3H]DFP-labeled proteins with different mol. wts, which disappeared on pretreatment of cells with cold DFP. However, stimulation of DFP-pretreated PMN with Ag-Ab complex in the presence of [3H]DFP resulted in the appearance of a [3H]DFP-labeled, membrane-bound protein with a mol. wt of 40,000. This protein was isolated by affinity chromatography of the solubilized PMN and phagosomes on anti-Ig antibody-Sepharose 4B. Although the enzymatic properties of the protein are not clear, the results so far obtained suggest that it is a putative, stimulus-activated serine protease participating in the triggering events leading to the activation of NADPH oxidase responsible for the respiratory burst and the formation of phagosomes.
D000818:Animals; D000936:Antigen-Antibody Complex / Q000276:immunology*; D004591:Electrophoresis, Polyacrylamide Gel; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007531:Isoflurophate / Q000494:pharmacology*; D008970:Molecular Weight; D009504:Neutrophils / Q000187:drug effects / Q000276:immunology*; D010047:Ovalbumin / Q000276:immunology; D010587:Phagocytosis; D011506:Proteins / Q000302:isolation & purification*; D013481:Superoxides / Q000378:metabolism
D000936:Antigen-Antibody Complex; D011506:Proteins; D013481:Superoxides; D007531:Isoflurophate; D010047:Ovalbumin