alpha2Macroglobulin is an abundant blood protein, that may play a critical role in cell growth and regulation through its ability to interact with many proteases and growth factors. The long term goals of this project are to elucidate the mechanism of action of alpha2-macroglobulin and to understand the basis for its physiological importance in interacting with proteases and growth factors. The experiments detailed in the present proposal are aimed at testing two hypotheses:- 1. That the specificity and efficiency of trapping of proteases by alpha2- macroglobulin can be understood in terms of the structure and location of the bait region (the site of limited proteolysis) and the internal thiol ester. 2. That growth factors interact only with an activated form of alpha2- macroglobulin which results either from initial reaction with limiting amounts of protease or else is present at a small equilibrium concentration, which is normally overlooked in examination of stoichiometric reactions. To test these hypotheses it is proposed to pursue the following specific aims:- 1. To further localize the functionally important bait region an thiol ester groups within the alpha2-macroglobulin tetramer using spectroscopic methods. 2. To obtain as much structural information as possible about the bait region from human alpha2-macroglobulin before and after reaction with various proteases using 1H NMR spectroscopic methods. 3. To continue attempts to obtain crystals of native and modified forms of alpha2-macroglobulin suitable for X-ray crystallographic analysis. 4. To determine the conditions necessary for interaction of the growth factors bFGF, EGF, PDGF, and TGF-beta with alpha2-macroglobulin, and to characterize the stoichiometry of interaction, the nature of the interaction, and the state of the alpha2-macroglobulin.
