Our studies are directed toward determining the biochemical mechanism involved in degranulation (secretion) of granulocytes (P M N) which affect the adherent properties of P M N. Recently the cell surface phorbol diester receptor has been suggested to be a protein kinase C. Since P M N activation is associated with the active metabolism of inositide lipids leading to the formation of the 1,2 diacyglycerol, phosphatidic acid, and arachidonic acid, we suggest that these biochemical changes might be related to fundamental mechanisms involved in P M N secretion. We propose to focus on the function of protein kinases, particularly protein kinase C and their role in phosphorylation of proteins which might be associated with the secretory process. Protein kinase C which is activated by diacyglycerol, will be purified from rabbit P M N using classical and affinity purification techniques. Protein phosphorylation patterns in whose cells, subcellular organelles, and membrane fractions will be determined and correlated with the P M N secretory behavior. In order to understand P M N secretion at a subcellular level we will employ a model system suitable for quantitative analysis which will allow us to examine in detail some of the components involved in promoting adherence and fusion of lysosomal granules with phagosomes. We will evaluate the specific interaction of actin with granule membranes. We will explore the role of lipids and calmodulin in promoting fusion of granule membranes to phagosomes. We will categorize by two-dimensional gel electrophoresis similarities and differences between the plasmalemma and specific and primary granule membranes of P M N. We will identify specific granule constituents (such as lactoferrin) on the plasma membrane of cells previously activated by electroblotting. Since P M N lactoferrin contributes to the adhesive properties of the PMN we plan to determine whether P M N lactoferrin can be reduced by rendering animals severely iron deficient which in turn could affect the functional behavior of these cells. Finally, we will examine both the functional properties and cellular distributions of proteins and enzymes that affect the functional properties of the P M N in a patient with specific granule deficiency and in another whole P M N lack a surface glycoprotein. These lines of investigation should prove fruitful in revealing the underlying mechanisms of stimulus-response coupling in P M N which lead to secretion. These studies will have relevance not only to the basic understanding of cellular responses to external stimuli but also to the mechanisms by which inappropriate responses are mainfested in abnormally functioning P M N.
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