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M. E. Estevez, A. V. Kolesnikov, V. I. Govardovskii, R. K. Crouch, P. Ala-Laurila, M. C. Cornwall; The 9-Methyl Group of Retinal Is Critical for Rapid Meta II Decay in Salamander Red Cones. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5952.
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© ARVO (1962-2015); The Authors (2016-present)
Previous in vitro biochemical experiments have suggested that the 9-methyl group of the visual pigment chromophore, 11-cis retinal, sterically interacts with red cone opsin to cause efficient hydrolysis of the Schiff base linkage between retinal and opsin. In accordance with this, electrophysiological experiments have shown that the response recovery after bright light exposure is greatly retarded in salamander red cones whose chromophore was substituted by 11-cis retinal lacking the 9-methyl group. However, no direct measurements of Meta II decay have been performed on intact cones containing visual pigment lacking the 9-methyl group. The purpose of the experiments described here was to measure the kinetics of visual pigment photolysis in salamander red cones regenerated with 11-cis 9-demethyl retinal.
Retinae isolated from Ambystoma mexicanum were bleached and regenerated with 35 µM 11-cis 9-demethyl retinal suspended in Ringer solution containing 0.5% ethanol and 1% BSA. Single intact red cone photoreceptors were isolated from retinae and identified based on their absorbance spectra. The products of photolysis (i.e. metapigments I/II/III, retinal, and retinol) were followed using a high-speed dichroic microspectrophotometer. In addition, retinol production was measured by microfluorimetry.
Red cones were successfully regenerated with 11-cis 9-demethyl retinal. The absorbance spectrum of these cones was significantly blue shifted (max = 524 nm) in comparison to red cones either containing their native 11-cis retinal/dehydroretinal mixture (max = 580 - 606 nm) or regenerated with 11-cis retinal (max = 566 nm). The timecourse of Meta II decay of red cones containing 11-cis 9-demethyl retinal was approximately 6 times slower (τ = 22.3 s) than red cones containing 11-cis retinal (τ = 3.5 s). Consistent with retarded Meta II decay, retinol production was significantly slower in red cones lacking the 9-methyl group of retinal.
Together with previous electrophysiological experiments on 9-demethyl red cones, these studies indicate that the presence of the 9-methyl group of retinal is critical for both normal rapid meta II decay and for timely recovery of the cone photoresponse following exposure to bright light.
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