INTERACTION OF ATP AND LENS ALPHA-CRYSTALLIN CHARACTERIZED BY EQUILIBRIUM BINDING-STUDIES AND INTRINSIC TRYPTOPHAN FLUORESCENCE SPECTROSCOPY

alpha-Crystallin, the most prevalent protein in vertebrate lenses, is a high molecular weight aggregate composed of alpha A and alpha B subu nits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The fo llowing parameters were obtained from equilibrium binding studies cond ucted at 37 degrees C: binding sites per 400 kDa aggregate=10 and K-a= 8.1.10(3) M(-1); and an essentially identical K-a of 7.84.10(3) M(-1) and 22 binding sites were determined for a 850 kDa aggregate. The coop erativity parameter, alpha H, approximates unity which denotes that th e binding of ligand is at independent sites. Binding was not significa nt at 22 degrees C and was absent at 4 degrees C. The specificity of t he binding site for ATP was established by intrinsic tryptophan fluore scence spectroscopy. In the presence of increasing concentrations of A TP (0.05-0.3 mM) tryptophan fluorescence decreases in a concentration dependent manner to a minimum at 0.2 mM above which there is a non-lin ear response. Quenching of fluorescence was not evident with P-i, AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the h ighest concentration (0.30 mM). Modulation of both supramolecular orga nization and lens metabolism is predicted as a consequence of ATP/alph a-crystallin binding.