KINETICS OF RHODOPSIN PHOTOLYSIS INTERMEDIATES IN RETINAL ROD DISK MEMBRANES—I. TEMPERATURE DEPENDENCE OF LUMIRHODOPSIN AND METARHODOPSIN I KINETICS

Abstract— Kinetic measurements have been carried out on rhodopsin photolysis intermediates in retinal rod membrane suspensions on a millisecond time scale over a wide spectral range at 10, 20 and 36°C. To adequately account for the data we find that a three exponential fit is required at most wavelengths and temperatures investigated. The fastest component at 380, 420, 480, 515 and 540 nm is due to the lumirhodopsin → metarhodopsin I transition. The slowest process is not isochromic with the larger amplitude process found on the metarhodopsin I → metarhodopsin II time scale. The properties of the larger amplitude slow component are identical with the classical metarhodopsin I → metarhodopsin II process. Effects of various experimental conditions are discussed. It is shown that scattered light, in particular, can significantly affect the measured kinetics. For example, sonication, low salt and refractive index matching reduce light scattering and increase the contribution of the lumirhodopsin → metarhodopsin I reaction to the absorption transients. Care must also be taken in the analysis because the isosbestic wavelengths in the spectral transients are highly temperature dependent. For example, the lumirhodopsin–metarhodopsin I isosbestic is 490–500 nm at 10°C, 480–490 nm at 20°C and to the blue of 470 nm at 36°C. Activation energies of 77.8, 130.9 and 101.3 kJ/mol were found for the lumirhodopsin → metarhodopsin I, the metarhodopsin I → metarhodopsin II and the slow millisecond processes, respectively. All three processes contribute to the signals at lower temperatures. The amplitude of the slowest component decreases as the temperature is raised, and at physiological temperature its amplitude is essentially negligible compared to the metarhodopsin I → metarhodopsin II reaction. The lumirhodopsin → metarhodopsin I reaction makes a large contribution to the amplitude of the signals at most wavelengths observed from 380–540 nm, especially at physiological temperatures. At physiological temperatures the decay rates of lumirhodopsin and metarhodopsin I are within a factor of three of each other. Thus, lumirhodopsin decay may be much more important for visual transduction than suggested by low temperature studies. In contrast to reports of several other laboratories we have no evidence for kinetic complexity in the metarhodopsin I → metarhodopsin II reaction.