RESUMO
We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.
Assuntos
Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Visão Ocular/fisiologia , Animais , Anuros , Domínio Catalítico , GMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Eletrofisiologia , Ativação Enzimática/efeitos dos fármacos , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Luz , Modelos Biológicos , Diester Fosfórico Hidrolases/química , Diester Fosfórico Hidrolases/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Segmento Externo da Célula Bastonete/metabolismo , Segmento Externo da Célula Bastonete/ultraestrutura , Transducina/química , Transducina/metabolismo , Transducina/farmacologia , Visão Ocular/efeitos da radiaçãoRESUMO
Proteins of the regulators of G protein signaling (RGS) family modulate the duration of intracellular signaling by stimulating the GTPase activity of G protein alpha subunits. It has been established that the ninth member of the RGS family (RGS9) participates in accelerating the GTPase activity of the photoreceptor-specific G protein, transducin. This process is essential for timely inactivation of the phototransduction cascade during the recovery from a photoresponse. Here we report that functionally active RGS9 from vertebrate photoreceptors exists as a tight complex with the long splice variant of the G protein beta subunit (Gbeta5L). RGS9 and Gbeta5L also form a complex when coexpressed in cell culture. Our data are consistent with the recent observation that several RGS proteins, including RGS9, contain G protein gamma-subunit like domain that can mediate their association with Gbeta5 (Snow, B. E., Krumins, A. M., Brothers, G. M., Lee, S. F., Wall, M. A., Chung, S., Mangion, J., Arya, S., Gilman, A. G. & Siderovski, D. P. (1998) Proc. Natl. Acad. Sci. USA 95, 13307-13312). We report an example of such a complex whose cellular localization and function are clearly defined.