Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1981 Nov;78(11):6873–6877. doi: 10.1073/pnas.78.11.6873

Interactions between photoexcited rhodopsin and GTP-binding protein: kinetic and stoichiometric analyses from light-scattering changes.

H Kühn, N Bennett, M Michel-Villaz, M Chabre
PMCID: PMC349154  PMID: 6273893

Abstract

In rod outer segments, photoexcited rhodopsin (R*) activates a cyclic GMP phosphodiesterase through a sequence of reactions involving a GTP-binding protein. By measuring light-scattering changes above 700 nm, we have studied the kinetics and stoichiometry of the association of R* with this protein and of the dissociation of the complex upon GDP/GTP exchange. Two light-scattering signals were obtained upon photoexcitation of rhodopsin in bovine rod outer segment membranes as well as in a reconstituted system consisting of purified GTP-binding protein and washed disc membranes; both signals depended specifically on the presence of GTP-binding protein. A "binding signal" that was observed in the absence of gTP as an increase in turbidity became saturated when a number of rhodopsin molecules equal to the number of GTP-binding protein molecules present (congruent to 10% in rod outer segments) has been bleached, suggesting that the protein binds to R* in a 1:1 complex. A "dissociation signal" of opposite sign, observed in presence of GTP at greater than or equal to 1 microM, is half maximal at 0.04% bleaching and saturated at 0.5% bleaching; it is interpreted as reflecting the dissociation of GTP-binding protein-R* complexes after GDP/GTP exchange on the GTP-binding protein, one R* being able to interact sequentially with about 100 GTP-binding protein molecules. The early time course of the binding signal is faster than that of the dissociation signal, and both signals take place in the 100-msec range at 20 degrees C.

Full text

PDF
6873

Images in this article

6874Image
on p.6874

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Bignetti E., Cavaggioni A., Fasella P., Ottonello S., Rossi G. L. Light and GTP effects on the turbidity of frog visual membrane suspensions. Mol Cell Biochem. 1980 Apr 18;30(2):93–99. doi: 10.1007/BF00227923. [DOI] [PubMed] [Google Scholar]
  2. Bignetti E., Cavaggioni A., Sorbi R. T. Light-activated hydrolysis of GTP and cyclic GMP in the rod outer segments. J Physiol. 1978 Jun;279:55–69. doi: 10.1113/jphysiol.1978.sp012330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Eckstein F., Cassel D., Levkovitz H., Lowe M., Selinger Z. Guanosine 5'-O-(2-thiodiphosphate). An inhibitor of adenylate cyclase stimulation by guanine nucleotides and fluoride ions. J Biol Chem. 1979 Oct 10;254(19):9829–9834. [PubMed] [Google Scholar]
  4. Fung B. K., Hurley J. B., Stryer L. Flow of information in the light-triggered cyclic nucleotide cascade of vision. Proc Natl Acad Sci U S A. 1981 Jan;78(1):152–156. doi: 10.1073/pnas.78.1.152. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Godchaux W., 3rd, Zimmerman W. F. Membrane-dependent guanine nucleotide binding and GTPase activities of soluble protein from bovine rod cell outer segments. J Biol Chem. 1979 Aug 25;254(16):7874–7884. [PubMed] [Google Scholar]
  6. Harary H. H., Brown J. E., Pinto L. H. Rapid light-induced changes in near infrared transmission of rods in Bufo marinus. Science. 1978 Dec 8;202(4372):1083–1085. doi: 10.1126/science.102035. [DOI] [PubMed] [Google Scholar]
  7. Hofmann K. P., Uhl R., Hoffmann W., Kreutz W. Measurements on fast light-induced light-scattering and -absorption changes in outer segments of vertebrate light sensitive rod cells. Biophys Struct Mech. 1976 Apr 15;2(1):61–77. doi: 10.1007/BF00535653. [DOI] [PubMed] [Google Scholar]
  8. Kwok-Keung Fung B., Stryer L. Photolyzed rhodopsin catalyzes the exchange of GTP for bound GDP in retinal rod outer segments. Proc Natl Acad Sci U S A. 1980 May;77(5):2500–2504. doi: 10.1073/pnas.77.5.2500. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Kühn H., Hargrave P. A. Light-induced binding of guanosinetriphosphatase to bovine photoreceptor membranes: effect of limited proteolysis of the membranes. Biochemistry. 1981 Apr 28;20(9):2410–2417. doi: 10.1021/bi00512a007. [DOI] [PubMed] [Google Scholar]
  10. Kühn H. Light- and GTP-regulated interaction of GTPase and other proteins with bovine photoreceptor membranes. Nature. 1980 Feb 7;283(5747):587–589. doi: 10.1038/283587a0. [DOI] [PubMed] [Google Scholar]
  11. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  12. Wheeler G. L., Bitensky M. W. A light-activated GTPase in vertebrate photoreceptors: regulation of light-activated cyclic GMP phosphodiesterase. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4238–4242. doi: 10.1073/pnas.74.10.4238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Yee R., Liebman P. A. Light-activated phosphodiesterase of the rod outer segment. Kinetics and parameters of activation and deactivation. J Biol Chem. 1978 Dec 25;253(24):8902–8909. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES