The aims of this proposal grow out of recent results from our research on protein modifying reactions occurring in the lens upon elevation of Ca2+-ion concentration. Specifically, this application reflects the continued commitment of our laboratory to devote its specialized protein chemical resources and knowledge in regard to the functioning of the endogenous cross-linking enzyme: transglutaminase for the aging of human lens and the problem of cataract formation. Subjects for investigation are grouped under the following topics: 1. Analysis of acceptor/donor relationships for the transglutaminase-catalyzed cross-linking of proteins in human and bovine lens. a. Identification of acceptor and donor protein partners in whole lens homogenates. b. Separation of the labeled acceptor and donor substrates of transglutaminase from other lens proteins. Raising of MAb's to the isolated acceptors and donors. c. Sequencing of acceptor and donor domains in the transglutaminase-reactive lens proteins. 2. Examination of oligomeric and polymeric structures formed by transglutaminase in human and bovine lens. a. New inhibitors of transglutaminase. b. Comparison of the 50K protein found in lenses of old individuals with X beta(2) generated in vitro through transglutaminase. c. Role of spectrin as a transglutaminase substrate in forming polymers. 3. Does 'oxidative stress' influence reactivities of human lens proteins with transglutaminase, and vice versa would transglutaminase action render these more susceptible to oxidative changes? 4. The human lens transglutaminase. cDNA sequence. Novel approaches for isolating the enzyme and its regulator(s). 5. Is there a turnover of N[epsilon](gamma-glutamyl)lysine in human lens?
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