Fh. Grus et H. Stieve, KINETICS OF RHODOPSIN REGENERATION IN-SITU AND IN THE EXCISED MEDIAN EYE OF LIMULUS-POLYPHEMUS MEASURED USING ELECTROPHYSIOLOGICAL METHODS, Pflugers Archiv, 435(6), 1998, pp. 827-833
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.