Related Experiment Videos
Isolation of a protein labeled with diisopropyl fluorophosphate on stimulation of polymorphonuclear leukocytes with
Abstract:
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.
Insights
Diisopropyl fluorophosphate (DFP) inhibits immune cell functions, but its effects are reversible. A novel 40,000-mol. wt. protein is identified, potentially mediating cellular activation.
Area of Science:
- Immunology
- Cell Biology
- Biochemistry
Background:
- Diisopropyl fluorophosphate (DFP) is known to inhibit oxygen radical (O2-) generation and phagocytosis in guinea-pig polymorphonuclear leukocytes (PMN).
- These inhibitory effects are reversible upon removal of DFP, indicating a regulatory mechanism rather than cell death.
Purpose of the Study:
- To investigate the molecular targets of DFP in PMN and identify proteins involved in stimulus-induced cellular activation.
- To elucidate the role of specific proteins in the respiratory burst and phagosome formation.
Main Methods:
- Labeling of PMN with [3H]DFP, followed by cell solubilization and SDS-PAGE to identify DFP-binding proteins.
- Stimulation of DFP-pretreated PMN with an antigen-antibody complex in the presence of [3H]DFP.
- Isolation of a specific DFP-labeled protein using affinity chromatography.
Main Results:
- Several [3H]DFP-labeled proteins were detected in intact PMN, disappearing upon pre-incubation with unlabeled DFP.
- Stimulation of DFP-pretreated PMN revealed a new, membrane-bound [3H]DFP-labeled protein with a molecular weight of 40,000.
- This 40,000-mol. wt. protein was isolated and appears to be a serine protease involved in activating NADPH oxidase.
Conclusions:
- A stimulus-activated, membrane-bound serine protease (40,000 mol. wt.) is identified as a key player in PMN activation.
- This protein likely mediates the triggering of NADPH oxidase, leading to the respiratory burst and phagosome formation.