- The aim of this proposal is to characterize the biochemical properties of human chymase, a serine class proteolytic enzyme secreted from mast cells, and investigate its interactions with protease inhibitors termed serpins (serine protease inhibitors). Many homeostatic processes contributing to protection from injury and inflammation involve secretion of proteases from inflammatory cells, such as mast cells. Inhibition of proteases by other proteins (e.g., serpins), provides regulation so that proteolysis is limited to specific processes. Therefore, understanding proteases and their inhibitors is important to health and disease. Recent findings indicate that chymase has high and low pH forms with different catalytic and substrate recognition properties. These forms may be important to the function of chymase and will be further investigated. X-ray crystal structure determination, site-directed mutagenesis, and spectroscopic analyses will be employed to understand the unique structural features of chymase producing these forms. The interaction of chymase with heparin, a proteoglycan secreted from mast cells, also will be studied using the recombinant chymase. This interaction is considered to have a role in regulating catalytic properties of chymase secreted from mast cells. These investigations, it is hoped, will define the catalytic properties of chymase and factors effecting its physiological substrate specificity more completely. The mechanism by which serpins inhibit proteases remains unclear. The effect of pH on the interaction of chymase with two serpins differed in a manner which suggests that two different intermediate steps may be involved in regulating the rate of inhibition. Further analysis of these two interactions using rapid mixing, spectroscopy, solvent isotope effects, and in vitro mutagenesis of chymase and serpin, will be utilized to define the biochemical nature (chemical or conformational) of these steps. These studies are aimed at establishing a more comprehensive mechanism for inhibition of proteases by serpins. It is hoped that these studies will advance our understanding of two classes of molecules which are important to production and regulation of inflammation.
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