Section 6
Chapter 5,721

Interaction between photo excited rhod opsin and peripheral enzymes in frog rana esculenta retinal rods influence on the post meta rhod opsin ii decay and phosphorylation rate of rhod opsin

Pfister, C.; Kuehn, H.; Chabre, M.

European Journal of Biochemistry 136(3): 489-500


ISSN/ISBN: 0014-2956
Accession: 005720656

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The major peripheral and soluble proteins in frog rod outer segment [ROS] preparations, and their interactions with photoexcited rhodopsin, were compared to those in cattle rod outer segments; they were similar. In particular the GTP-binding protein (G) has the same subunit composition, the same abundance relative to rhodopsin (1/10) and it undergoes the same light and nucleotide-dependent interactions with rhodopsin in both preparations. Previous work on cattle ROS has shown that photoexcited rhodopsin (R), in a state identified with metarhodopsin II, associates with the G protein as a first step to the light-activated GDP/GTP exchange on G. The complex R.sbd.G is stable in absence of GTP, but is rapidly dissociated by GTP owing to the GDP/GTP exchange reaction. Low bleaching extents (< 10% R) in absence of GTP therefore create predominantly R.sbd.G complexes, whereas bleaching in presence of GTP creates free R. Under conditions of complexed R, 2 reactions of R in frog ROS are highly perturbed as compared to free R: the spectral decay of metarhodopsin II (MII) into later photoproducts, and the phosphorylation of R by an ATP-dependent protein kinase. Spectral measurements were performed using linear dichroism on oriented frog ROS; this technique allows discrimination between MII and later photoproducts absorbing at the same wavelength. Association of R with G leads to a strong reduction of the amount of MIII formed and to an acceleration of the decay of MIII. MII is significantly stabilized, in agreement with the hypothesis that MII is the intermediate which binds to G. Phosphorylation of R was strongly inhibited under conditions of R.sbd.G complex formation as compared to free R. Interferences between reactions at the 3 sites involved in R are discussed: the retinal binding site in the hydrophobic core is sensitive to the presence of GTP-binding protein at its binding site on the cytoplasmic surface of R; the kinase and the GTP-binding protein compete for access to their respective binding sites, both located on the surface of R. A slow and nucleotide-dependent light-induced binding of a protein of MW 50,000 was observed, which was considered as the equivalent of the 48,000 MW light-dependent protein previously identified in cattle ROS.

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