ウシ視細胞桿体外節の視興奮過程におけるロドプシソとGTP結合蛋白質(トランスデューシン)およびアレスチンの相互作用
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In bovine rod outer segments (ROS), transducin (T), a GTP-binding protein, couples a photobleaching intermediate of rhodopsin, metarhodopsin II (meta Rh II), to an activation of cGMP-phosphodiesterase (PDE) with a remarkable amplification (ON-process). Activated PDE is subsequently deactivated by a phosphorylation of meta Rh II in the presence of arrestin (OFF-process). To analyze the mechanism by which phosphorylated meta Rh II and arrestin (Arr) deactivate the PDE, each subunit component of T (Tα and Tβγ) and Arr was purified. We used thoroughly washed ROS membranes, whose Rh had been previously phosphorylated (P-disk), and to which purified T and Arr were reconstituted. The results were compared with those obtained with a C-disk, which was similarly prepared to the P-disk except that the Rh was not phosphorylited. Purified Tβγ was separated into two components, i.e., Tβγ-1 and Tβγ-2, by an anion exchange Mono Q column chromatography. In the presence of meta Rh II in the P-disk or the C-disk, GppNHp (nonhydrolyzable analogue of GTP) binding to Ta was remakably enhanced with an increase in concen?tration of Tβγ-2. On the contrary, Tβγ-1 retained little, if any, to enhance the binding. Phosphorylation of Rh gave no influence of GppNHp binding to Tα in the presence of Tβγ-1 or Tβγ-2. The difference in the ability to enhance the GppNHp binding to Tα between the two components was attributed to the different affinities for meta Rh II. That is, Tα, Tβγ-2 and Rh in the C-disk or the P-disk associated to form a transient trimeric complex only when Rh transformed into meta Rh II upon photon absorption. Addition of GppNHp dissociated both Tα and Tβγ-2 from meta Rh II. When Tβγ-2 was replaced with Tβγ-1 in this experiment, the formation of the trimeric complex was scarcely observed. In contrast, Arr associated with meta Rh II only when Rh had been previously phosphorylated (P-disk), and the addition of GppNHp had no effect on the association. Interestingly, the association of Arr with meta Rh II in the P-disk did not inhibit the co-association of Tα and Tβγ-2 with the meta Rh II. These results suggest that Rh phosphorylation and Arr supress PDE activity directly rather than by competitive inhibitions upon the binding of T to phosphorylated meta Rh II by Arr.
- 札幌医科大学の論文
- 1988-08-01
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