Über Primärreaktionen beim Sehvorgang. Thermodynamischer und kinetischer Einfluß des pH-Wertes auf die Metarhodopsin-I-II-Umwandlung Protonenverbrauch als Auswirkung einer Konform ationsänderung / Primary Reactions in the Visual Process Thermodynamic and Kinetic Influence of pH on the Metarhodopsin I-II Transition Proton Consumption as an Effect of a Conformation Change

Abstract

Abstract In isolated bovine rod outer segments metarhodopsin I -II absorption-changes are measured using rapid flash-photometry. The rapid H+-uptake accompanying this transition is measured using phthaleine pH-indicators. 1. The meta I -II equilibrium is pH-dependent in the same manner as the proton uptake (Figs 1 ,1 4). This can be explained by the assumption that the meta II conformation is stabilized by the protonization of an amino-group, which in its protonized state cannot be re-enfolded into the hydrophobic core of the molecule, since it is too polar (Fig. 15). In the proposed model the con­ formation-regulating proton binding group is not in the chromophoric region of rhodopsin. This resolves the well known contradiction between the expected bathochromic and observed hypso-chromic shift of meta II H+. The model furthermore involves that the dissociation-constant of the protonized group differs from the measured apparent pK = 6.3. 2. The meta I -II transition is H+ (and OH-)-catalysed (Figs 4, 5). The kinetics show usually a characteristic deviation from a first-order process (Fig. 2). This can be explained by some in­ homogeneity of the rhodopsin-molecules e. g. some differences in the shieldings of the catalytic centers from H+ (and OH-). 3. The activation energy decreases with increasing proton-concentration (Fig. 6). The protons seem to open a new reaction pathway with a smaller activation energy. 4. Triton X 100 and ethanol increase, sodium-desoxycholate slows down the velocity of the M-I-II-transition, whereas digitonin has only a small effect (Fig. 7). 5. The relaxation-velocity of the optical pH-signal depends on the structural state of the rhodopsin-containing substrate (Figs 10. 11). The outer membrane of the outer segment and the disc-membrane are regarded as possible H+-diffusion-barriers (Fig. 12), because the time lag between MII-signal and pH-signal is nearly eliminated, when the membranes are destroyed by digitonin. 6. In some experiments, using successive non-saturating flashes, an initial H+-uptake of 2 - 3 H+/MII was observed (Fig. 17). After full-bleaching, however, the ΣH+/ΣMII-ratio is one. The hypothesis of a conformative coupling of 2 - 3 rhodopsin-molecules is presented (Fig. 18). The possible existence of an "unfolded" state of rhodopsin is discussed on the basis of a photo-regeneration-experiment, in which no H+-release was observed (Figs 19, 20)