Ligand binding to the adenine analogue binding protein of the rabbit erythrocyte.

Adenine analogue binding protein of rabbit erythrocytes reversibly binds [3H] adenosine with a KD of 5.3 X 10(-9) M, an association rate constant of 1.4 X 10(-12) M-1 min-1 and a dissociation rate constant of 7.5 X 10(-3) min-1, as estimated by a nonlinear curve-fitting program applied to data on the time course of the binding reaction. Independent estimates of KD by Scatchard plots and of the dissociation rate constant by dilution or adsorption of free [3H] adenosine on charcoal or by the addition of excess adenosine agreed closely with the estimates from the curve-fitting program. Inhibition of [3H] adenosine binding by a series of 77 adenosine analogues was used to define the factors determining the binding affinity of this nucleoside. These are: (1) the size and aromaticity of the purine base; (2) a glycosylic torsion angle of approximately -120 degrees; (3) the ribo configuration of the 2'-and 3'-hydroxyls and also the 5'-hydroxyl. Bulky substituents in the region of C-2' and to a lesser extent in the region of C-3' reduce binding affinity.