The FIRST award application is designed to characterize the biochemical and functional aspects of a defined human neutrophil (PMN) surface antigen which is involved in cellular activation. Whereas the dynamics of neutrophil effector responses to chemoattractant are well studied, little is known about the molecular mechanisms which translate PMN stimulus exposure into activation of cell microbicidal responses. The proposed research uses a function-inhibiting PMN-specific monoclonal antibody, called Ab 1-15, to probe the involvement of a 68,000 MW PMN surface molecule in metabolic pathways of PMN activation. Ab 1-15 inhibits PMN secretory and adhesive responses to the bacterial peptide Nformyl-met-leu-phe (FMLP) but does not affect chemotactic response to this agent. Furthermore, Ab 1-15 inhibits a chymotrypsin-like protease activity localized in purified PMN plasma membrane. The experiments described in this project are designed to characterize the proteolytic activity of the 68,000 MW Ab 1-15 antigen and define its relative enzymatic affinity for FMLP ligand and for function-related molecules within the PMN membrane. The ability of chymotryptic proteases to modify the function of intact PMN and isolated cell membrane will also be assessed: studies of chymotrypsin's effects on PMN membrane metabolism will determine the effects of chymotryptic proteolysis on mechanisms of superoxide anion generation, cation exchange, polyphosphoinositide release, and release of arachadonic acid. In addition, studies will define the ability of chymotryptic enzymes to modify the inhibitory nucleotide regulatory protein, the Ni molecule, involved both in PMN activation and in regulation of cyclic AMP metabolism. These studies will be correlated with analysis of the effects of protease treatment on defined, membrane-related components (e.g. receptor molecules, Ns and Ni molecules, and actin binding protein), thus providing insight into potential function-related modification of specific PMN membrane molecules. These experiments, which allow functional and structural identification of the sites of action of a function- related Ab 115 antigen/protease will provide needed understanding of the biochemical events involved in early, FMLP receptor-mediated, membrane activation.
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