This continuation application examines two Ca2+ triggered post-translational enzymatic reactions of remodeling of lens proteins which contribute to opacification and possibly to cataract formation. Thus, specific aims relate to basic research, as well as to pathological applications, and are subdivided into the following categories: 1) covalent structural changes affecting proteins of the lens upon enrichmenbt with Ca2 ions will be analyzed in terms of proteolysis and cross-linking in Ca2+ treated lens; testing of inhibitors of transglutaminase-mediated cross-linking of both the competitive and noncompetitive type; identification of protein targetw which are susceptible to proteolysis or cross-linking by transglutaminase in Ca2+ treated lens will be identified by enzyme-dependent, site-specific labelling with amines and also by immunological analysis of epitopes shared between mmonomeric protein substrates and the cross-linked polymers. 2) Properties and regulation of transglutaminase, including further purification of the enzyme; immunological studies with lens transglutaminase, kinetic appraisal of lens transglutaminase; effects of activators and inhibitors of lens transglutaminase. 3) The Gamma-glutamyl-Xi-lysine cross-link content of the 55,000-57,000 weight doublet band generated in Ca2+ treated rabbit lens. 4) Relevance of the protein changes found in Ca2+ treated lens to problems of animal and human cataract. 5) Frequency of Gamma-glutamyl-Xi-lysine cross-links in the non-reducible high molecular weight polymers of human cataract will be examined.
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