THE KINETICS AND THERMODYNAMICS OF BLEACHING OF RHODOPSIN IN DIMYRISTOYLPHOSPHATIDYLCHOLINE - IDENTIFICATION OF META-I, META-II, AND META-III INTERMEDIATES

The effects of light on rhodopsin reconstituted into dimyristoylphosph atidylcholine at a molar ratio of 1:70 have been studied as a function of temperature and time. The lipid phase behavior and thermal stabili ty of rhodopsin in the system used to measure the photolytic reactions were also determined. Thus, it was shown that the gel-to-fluid phase transition of the reconstituted membrane had a marked influence on the bleaching kinetics and thermodynamics of rhodopsin-bleaching equilibr ia, whereas lipid-protein interactions were also directly involved. Rh odopsin photolysis resulted in temperature-sensitive equilibria betwee n three main photoproducts, with absorption maxima of approximately 48 0, 380, and 465 nm. Below the lipid phase transition temperature, the main photoproduct had an absorption maximum at 480 nm. With increasing temperature progressively more of the 380 nm-absorbing species was fo rmed. The photoproduct with a spectral-maximum at 465 nm absorption wa s formed more slowly. Increasing temperatures decreased the ratio of t he 465:380 nm-absorbing species. The thermal reactions were reversible : on cooling the higher-temperature products were converted back to th e lower-temperature products. The results indicate that rhodopsin has extensive photochemical activity when reconstituted in dimyrisotylphos phatidylcholine. The equilibria that we have measured resemble those o f rhodopsin in the disk membrane. However, the kinetics of meta-II and meta-III formation appear to be considerably faster in the reconstitu ted membranes and the meta-I-to-meta-II equilibrium is displaced in th e direction of the meta-I state relative to native rod outer segment d isk membranes. The displacement of the meta-rhodopsin equilibrium from its position in the rod outer segment is attributed mainly to the eff ects of lipid-lipid interactions in the membrane bilayer and correlate s with the difference in gel-to-fluid phase transition temperature of the different lipids.