Our studies are directed toward determining the biochemical mechanisms involved in degranulation (secretion) of granulocytes (PMN) which affect the adherent properties of PMN. We propose to focus on protein kinase C and its role in phosphorylation of proteins which might be associated with the secretory process. Protein kinase C will be purified from human PMN using high performance liquid chromatography (HPLC). Protein phosphorylation patterns in whole cells, subcellular organelles, and membrane fractions will be determined and correlated with the PMN secretroy behavior. We will purify an endogenous inhibitor of PKC localized in specific granules of PMN. The inhibitor will be characterized with respect to its specific intracellular localization, molecular weight, and activity as a regulator of PKC. To understand PMN secretion at a subcellular level we will employ a model system suitable for quantitative analysis which will allow us to examine in detail the interrelationship between PKC binding to PMN membranes and subsequent functional effects. We will examine in detail some of the components involved in promoting the adherence and fusion of granules with membranes. We will evaluate the specific interaction of actin with granule membranes. We will explore the role of lipids, calmodulin, protein kinase C in promoting fusion of granule membranes to phagosomes and to plasma membranes. We will categorize by 2-dimensional gel electrophoresis similarities and differences between the plasma membrane and specific and primary granules and secretory vesicles of PMN. We will identify intracellular organelle constituents such as lactoferrin and C3bi on the plasma membrane of cells previously activated by electroblotting. We will examine the biochemical mechanisms underlying the hyperresponsive behavior of specific granule deficient PMNs which are unable to adhere normally to surfaces. We will also examine by pulse-chase experiments the synthesis of adhesive glycoproteins and their accumulation on the plasma membrane of normal and deficient canine monocytes.
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