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. 1991 Apr;10(4):793–798. doi: 10.1002/j.1460-2075.1991.tb08011.x

A 26 kd calcium binding protein from bovine rod outer segments as modulator of photoreceptor guanylate cyclase.

H G Lambrecht 1, K W Koch 1
PMCID: PMC452718  PMID: 1672637

Abstract

The resynthesis of cGMP in vertebrate photoreceptors by guanylate cyclase is one of the key events leading to the reopening of cGMP-gated channels after photoexcitation. Guanylate cyclase activity in vertebrate rod outer segments is dependent on the free calcium concentration. The basal activity of the enzyme observed at high concentrations of free calcium (greater than 0.5 microM) increases when the free calcium concentration is lowered into the nanomolar range (less than 0.1 microM). This effect of calcium is known to be mediated by a soluble calcium-sensitive protein in a highly cooperative way. We here show that this soluble protein, i.e. the modulator of photoreceptor guanylate cyclase, is a 26 kd protein. Reconstitution of the purified 26 kd protein with washed rod outer segment membranes containing guanylate cyclase revealed a 3- to 4-fold increase of cyclase activity when the free calcium concentration was lowered in the physiological range from 0.5 microM to 4 nM. Guanylate cyclase in whole rod outer segments was stimulated 10-fold in the same calcium range. The activation process in the reconstituted system was similar to the one in the native rod outer segment preparation, it showed a high cooperativity with a Hill coefficient n between 1.4 and 3.5. The half-maximal activation occurred between 110 and 220 nM free calcium. The molar ratio of the modulator to rhodopsin is 1:76 +/- 32. The protein is a calcium binding protein as detected with 45Ca autoradiography. Partial amino acid sequence analysis revealed a 60% homology to visinin from chicken cones.

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Selected References

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

  1. Ames A., 3rd, Walseth T. F., Heyman R. A., Barad M., Graeff R. M., Goldberg N. D. Light-induced increases in cGMP metabolic flux correspond with electrical responses of photoreceptors. J Biol Chem. 1986 Oct 5;261(28):13034–13042. [PubMed] [Google Scholar]
  2. Barkdoll A. E., 3rd, Pugh E. N., Jr, Sitaramayya A. Calcium dependence of the activation and inactivation kinetics of the light-activated phosphodiesterase of retinal rods. J Gen Physiol. 1989 Jun;93(6):1091–1108. doi: 10.1085/jgp.93.6.1091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Burgess W. H., Jemiolo D. K., Kretsinger R. H. Interaction of calcium and calmodulin in the presence of sodium dodecyl sulfate. Biochim Biophys Acta. 1980 Jun 26;623(2):257–270. doi: 10.1016/0005-2795(80)90254-8. [DOI] [PubMed] [Google Scholar]
  5. Cervetto L., Lagnado L., Perry R. J., Robinson D. W., McNaughton P. A. Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients. Nature. 1989 Feb 23;337(6209):740–743. doi: 10.1038/337740a0. [DOI] [PubMed] [Google Scholar]
  6. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol. 1988;157:378–417. doi: 10.1016/0076-6879(88)57093-3. [DOI] [PubMed] [Google Scholar]
  7. Fain G. L., Matthews H. R. Calcium and the mechanism of light adaptation in vertebrate photoreceptors. Trends Neurosci. 1990 Sep;13(9):378–384. doi: 10.1016/0166-2236(90)90023-4. [DOI] [PubMed] [Google Scholar]
  8. Forti S., Menini A., Rispoli G., Torre V. Kinetics of phototransduction in retinal rods of the newt Triturus cristatus. J Physiol. 1989 Dec;419:265–295. doi: 10.1113/jphysiol.1989.sp017873. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Grynkiewicz G., Poenie M., Tsien R. Y. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem. 1985 Mar 25;260(6):3440–3450. [PubMed] [Google Scholar]
  10. Hodgkin A. L., Nunn B. J. Control of light-sensitive current in salamander rods. J Physiol. 1988 Sep;403:439–471. doi: 10.1113/jphysiol.1988.sp017258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Huppertz B., Weyand I., Bauer P. J. Ca2+ binding capacity of cytoplasmic proteins from rod photoreceptors is mainly due to arrestin. J Biol Chem. 1990 Jun 5;265(16):9470–9475. [PubMed] [Google Scholar]
  12. Kaupp U. B., Hanke W., Simmoteit R., Lühring H. Electrical and biochemical properties of the cGMP-gated cation channel from rod photoreceptors. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 1):407–415. doi: 10.1101/sqb.1988.053.01.048. [DOI] [PubMed] [Google Scholar]
  13. Kawamura S., Murakami M. Regulation of cGMP levels by guanylate cyclase in truncated frog rod outer segments. J Gen Physiol. 1989 Oct;94(4):649–668. doi: 10.1085/jgp.94.4.649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Koch K. W., Eckstein F., Stryer L. Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments. J Biol Chem. 1990 Jun 15;265(17):9659–9663. [PubMed] [Google Scholar]
  15. Koch K. W., Stryer L. Highly cooperative feedback control of retinal rod guanylate cyclase by calcium ions. Nature. 1988 Jul 7;334(6177):64–66. doi: 10.1038/334064a0. [DOI] [PubMed] [Google Scholar]
  16. Kondo H., Miller W. H. Rod light adaptation may be mediated by acceleration of the phosphodiesterase-guanylate cyclase cycle. Proc Natl Acad Sci U S A. 1988 Feb;85(4):1322–1326. doi: 10.1073/pnas.85.4.1322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Korenbrot J. I., Miller D. L. Cytoplasmic free calcium concentration in dark-adapted retinal rod outer segments. Vision Res. 1989;29(8):939–948. doi: 10.1016/0042-6989(89)90108-9. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. 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]
  20. Maruyama K., Mikawa T., Ebashi S. Detection of calcium binding proteins by 45Ca autoradiography on nitrocellulose membrane after sodium dodecyl sulfate gel electrophoresis. J Biochem. 1984 Feb;95(2):511–519. doi: 10.1093/oxfordjournals.jbchem.a134633. [DOI] [PubMed] [Google Scholar]
  21. Matthews H. R., Fain G. L., Murphy R. L., Lamb T. D. Light adaptation in cone photoreceptors of the salamander: a role for cytoplasmic calcium. J Physiol. 1990 Jan;420:447–469. doi: 10.1113/jphysiol.1990.sp017922. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Matthews H. R., Murphy R. L., Fain G. L., Lamb T. D. Photoreceptor light adaptation is mediated by cytoplasmic calcium concentration. Nature. 1988 Jul 7;334(6177):67–69. doi: 10.1038/334067a0. [DOI] [PubMed] [Google Scholar]
  23. Meyer T., Wensel T., Stryer L. Kinetics of calcium channel opening by inositol 1,4,5-trisphosphate. Biochemistry. 1990 Jan 9;29(1):32–37. doi: 10.1021/bi00453a004. [DOI] [PubMed] [Google Scholar]
  24. Minta A., Kao J. P., Tsien R. Y. Fluorescent indicators for cytosolic calcium based on rhodamine and fluorescein chromophores. J Biol Chem. 1989 May 15;264(14):8171–8178. [PubMed] [Google Scholar]
  25. Nakatani K., Yau K. W. Calcium and light adaptation in retinal rods and cones. Nature. 1988 Jul 7;334(6177):69–71. doi: 10.1038/334069a0. [DOI] [PubMed] [Google Scholar]
  26. Nakatani K., Yau K. W. Sodium-dependent calcium extrusion and sensitivity regulation in retinal cones of the salamander. J Physiol. 1989 Feb;409:525–548. doi: 10.1113/jphysiol.1989.sp017511. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pepe I. M., Panfoli I., Cugnoli C. Guanylate cyclase in rod outer segments of the toad retina. Effect of light and Ca2+. FEBS Lett. 1986 Jul 14;203(1):73–76. doi: 10.1016/0014-5793(86)81439-9. [DOI] [PubMed] [Google Scholar]
  28. Ratto G. M., Payne R., Owen W. G., Tsien R. Y. The concentration of cytosolic free calcium in vertebrate rod outer segments measured with fura-2. J Neurosci. 1988 Sep;8(9):3240–3246. doi: 10.1523/JNEUROSCI.08-09-03240.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rogers J. H. Calretinin: a gene for a novel calcium-binding protein expressed principally in neurons. J Cell Biol. 1987 Sep;105(3):1343–1353. doi: 10.1083/jcb.105.3.1343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Schnetkamp P. P., Daemen F. J. Isolation and characterization of osmotically sealed bovine rod outer segments. Methods Enzymol. 1982;81:110–116. doi: 10.1016/s0076-6879(82)81019-7. [DOI] [PubMed] [Google Scholar]
  31. Stryer L. Molecular basis of visual excitation. Cold Spring Harb Symp Quant Biol. 1988;53(Pt 1):283–294. doi: 10.1101/sqb.1988.053.01.035. [DOI] [PubMed] [Google Scholar]
  32. Torre V., Matthews H. R., Lamb T. D. Role of calcium in regulating the cyclic GMP cascade of phototransduction in retinal rods. Proc Natl Acad Sci U S A. 1986 Sep;83(18):7109–7113. doi: 10.1073/pnas.83.18.7109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vuong T. M., Chabre M. Subsecond deactivation of transducin by endogenous GTP hydrolysis. Nature. 1990 Jul 5;346(6279):71–74. doi: 10.1038/346071a0. [DOI] [PubMed] [Google Scholar]
  34. Yamagata K., Goto K., Kuo C. H., Kondo H., Miki N. Visinin: a novel calcium binding protein expressed in retinal cone cells. Neuron. 1990 Mar;4(3):469–476. doi: 10.1016/0896-6273(90)90059-o. [DOI] [PubMed] [Google Scholar]
  35. Yau K. W., Baylor D. A. Cyclic GMP-activated conductance of retinal photoreceptor cells. Annu Rev Neurosci. 1989;12:289–327. doi: 10.1146/annurev.ne.12.030189.001445. [DOI] [PubMed] [Google Scholar]
  36. Yau K. W., Nakatani K. Light-induced reduction of cytoplasmic free calcium in retinal rod outer segment. Nature. 1985 Feb 14;313(6003):579–582. doi: 10.1038/313579a0. [DOI] [PubMed] [Google Scholar]

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