The aim of this research is to extend our understanding of the biochemical basis of inflammation, and of certain interactions between the inflammatory and immune responses. In the renewal application, we will continue to approach this goal by the design and synthesis of peptides which can serve as specific substrates and inhibitors of the C3/5 convertases, key enzymes in complement-mediated inflammation. Peptides are synthesized by the Merrifield method of solid-phase synthesis, and are derivitized in solution. Substrate activity is typically measured by th rlease of a fluoreschent molecule, such as amino methyl coumarin, upon enzymatic cleavage. Due to certain limitations of this method, however, we also intend to develop a new type of assay for proteases which enables us to include amino acids on both sides of the cleavage site in our synthetic substrates. For inhibiroty peptides, we plan to focus our attention on the use of chloromethyl ketone derivatives of peptides which have proven to be specific substrates. Due to their central position in the complement cascade, we will focus our attention on the C3/5 convertases. As we are most interested in the ability of our substrates and inhibitors to affect these convertases selectively in a complex mixture of enzymes such as serum, we will carefully define the specificity of these peptides in such systems. Peptides substrates will then be used to assay the complement enzyme in such mixtures; peptide inhibitors will be used to block complement selectively to define the effect of such inhibition on phenomenon which involve the interaction of different types of cells and of plasma. Our ability to manipulate these interactions by the use of peptide inhibitors will be evaluated.
