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 Ka = 8.1 x 10(3) M-1; and an essentially identical Ka of 7.84 x 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 of 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.
Palmisano
D V
DV
Department of Physiology, UMD-New Jersey Medical School, Newark 07103.
Groth-Vasselli
B
B
Farnsworth
P N
PN
Reddy
M C
MC
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D003459:Crystallins / Q000378:metabolism*; D006160:Guanosine Triphosphate / Q000378:metabolism / Q000494:pharmacology; D007908:Lens, Crystalline / Q000737:chemistry*; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D008970:Molecular Weight; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry*
D003459:Crystallins; D046911:Macromolecular Substances; D006160:Guanosine Triphosphate; D014364:Tryptophan; D000255:Adenosine Triphosphate