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 subunits. Evidence is presented that ATP, a major phosphorus metabolite of the lens binds to alpha-crystallin extracted from calf lenses. The following parameters were obtained from equilibrium binding studies conducted 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 cooperativity parameter, alpha H, approximates unity which denotes that the binding of ligand is at independent sites. Binding was not significant at 22 degrees C and was absent at 4 degrees C. The specificity of the binding site for ATP was established by intrinsic tryptophan fluorescence spectroscopy. In the presence of increasing concentrations of ATP (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-linear response. Quenching of fluorescence was not evident with P-i, AMP or ADP. GTP elicited a minimal quenching of fluorescence only at the highest concentration (0.30 mM). Modulation of both supramolecular organization and lens metabolism is predicted as a consequence of ATP/alpha-crystallin binding.