Effect of digitonin concentration on regeneration of cattle rhodopsin

Abstract

The effect of digitonin concentration on the regeneration of cattle rhodopsin was studied. The second-order rate constant ( k 2) of the regeneration of rhodopsin was measured under pseudo first-order conditions of 11- cis-retinal excess in the presence of digitonin at various concentrations, showing that above the critical micelle concentration of digitonin the regeneration is inhibited more than that below the critical micelle concentration. Far below the critical micelle concentration (<0.01%), k 2 reaches a constant value, 7500 M −1 · s −1, at 25°C and pH 7.0. Far above the critical micelle concentration (2%), k 2 is 50 M −1 · s −1, which agrees with that reported previously by Wald and Brown (Wald, G. and Brown, P.K. (1956) Nature 177, 174–176). The regeneration of rhodopsin in rod outer segments was also measured, showing that k 2 in rod outer segments is similar (5600 M −1 · s −1) to that in digitonin below the critical micelle concentration. The inhibition of the regeneration of rhodopsin by β-ionone (8.71 · 10 −5 M) diminishes above the critical micelle concentration of digitonin. The activation energies of the regeneration of rhodopsin above and below the critical micelle concentration of digitonin are approx. 22 kcal/mol. These results suggest that the dissociation constant of an 11- cis-retinal · opsin complex ( K m), in which 11- cis-retinal and opsin are postulated to bind each other through a β-ionone ring-binding site in opsin, increases above the critical micelle concentration of digitonin more than that below the critical micelle concentration, and that under both conditions the rate-determining step is the slow formation process of the retinylidene-protonated Schiff base in rhodopsin. Other activation parameters were estimated on the regeneration of rhodopsin above and below the critical micelle concentration of digitonin, which were discussed in the light of the above-mentioned mechanism. The dark adaptation process in vivo, which reflects the regeneration of rhodopsin, was discussed, and an existence of a pool of a precursor of 11- cis-retinal was suggested.