KINETICS OF RHODOPSIN REGENERATION IN-SITU AND IN THE EXCISED MEDIAN EYE OF LIMULUS-POLYPHEMUS MEASURED USING ELECTROPHYSIOLOGICAL METHODS

The reactivation of rhodopsin after photoregeneration from metarhodops in in the UV-sensitive cells of the median eye of Limulus was examined by means of extracellular electroretinogram (ERG) measurements. Absor bed photons convert the transducing rhodopsin (Rt) to metarhodopsin, w hich is thermostable and can be reconverted by another photon to non-t ransducing rhodopsin (Rn). The amplitude of the ERG is assumed to corr elate linearly with the amount of Rt under otherwise constant conditio ns. The results demonstrate that the reactivation of Rn recorded in vi vo in the intact animal is much faster than that in the excised eye [h alf period: 4 min (in vivo), 23 min (excised eye)]. In the excised eye the ERC amplitudes recover over a sigmoidal time course; however, in vivo the kinetics often appear to be exponential. The in vivo kinetics were measured by several defined molar ratios of Rn to rhodopsin plus metarhodopsin [Rn/(R + M)]. These were adjusted by different duration s of pre-illumination with UV light, which is preferentially absorbed by rhodopsin. The in vivo kinetics ics were fitted by a single exponen tial function. At very high molar ratios of Rn (> 90%) the kinetics be come sigmoidal even in the in vivo experiments. The half-life of the i n vivo kinetics depends linearly on the initial molar Rn/(R + M) ratio [half-life: 4-12 min with a rise of 0.1 min/% inactivated metarhodops in (Mn), 20 degrees C]. The results are consistent with the assumption of multiple-step dephosphorylation being the I rate-limiting step in regeneration of rhodopsin in the dark.