Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1998 Mar 15;330(Pt 3):1201–1208. doi: 10.1042/bj3301201

Large-scale production and purification of the human green cone pigment: characterization of late photo-intermediates.

P M Vissers 1, P H Bovee-Geurts 1, M D Portier 1, C H Klaassen 1, W J Degrip 1
PMCID: PMC1219262  PMID: 9494086

Abstract

We present the first characterization of the late photo-intermediates (Meta I, Meta II and Meta III) of a vertebrate cone pigment in a lipid environment. Marked differences from the same pathway in the rod pigment were observed. The histidine-tagged human green cone pigment was functionally expressed in large-scale suspension cultures in Sf9 insect cells using recombinant baculovirus. The recombinant pigment was extensively purified in a single step by immobilized metal affinity chromatography and displays the expected spectral characteristics. The purified pigment was able to activate the rod G-protein transducin at about half the rate of the rod pigment. Following reconstitution into bovine retina lipid proteoliposomes, identification and analysis of the photo-intermediates Meta I, Meta II and Meta III was accomplished. Similar to the rod pigment, our results indicate the existence of a Meta I-Meta II equilibrium, but we find no evidence for pH dependence. Replacement of native Cl- by NO3- in the anion-binding site of the cone pigment affected the spectral position of the pigment itself and of the Meta I intermediate, but not that of Meta II and Meta III. The decay rate of the 'active' intermediate Meta II did not differ for the Cl- and NO3- state. However, in qualitative agreement with results reported before for chicken cone pigments, the rate of Meta II decay was significantly higher in the human cone pigment than in the rod pigment.

Full Text

The Full Text of this article is available as a PDF (359.1 KB).

Selected References

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

  1. Asenjo A. B., Rim J., Oprian D. D. Molecular determinants of human red/green color discrimination. Neuron. 1994 May;12(5):1131–1138. doi: 10.1016/0896-6273(94)90320-4. [DOI] [PubMed] [Google Scholar]
  2. Clark N. A., Rothschild K. J., Luippold D. A., Simon B. A. Surface-induced lamellar orientation of multilayer membrane arrays. Theoretical analysis and a new method with application to purple membrane fragments. Biophys J. 1980 Jul;31(1):65–96. doi: 10.1016/S0006-3495(80)85041-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. De Grip W. J., Daemen F. J., Bonting S. L. Isolation and purification of bovine rhodopsin. Methods Enzymol. 1980;67:301–320. doi: 10.1016/s0076-6879(80)67038-4. [DOI] [PubMed] [Google Scholar]
  4. DeCaluwé G. L., Bovee-Geurts P. H., Rath P., Rothschild K. J., de Grip W. J. Effect of carboxyl mutations on functional properties of bovine rhodopsin. Biophys Chem. 1995 Sep-Oct;56(1-2):79–87. doi: 10.1016/0301-4622(95)00018-s. [DOI] [PubMed] [Google Scholar]
  5. Delange F., Merkx M., Bovee-Geurts P. H., Pistorius A. M., Degrip W. J. Modulation of the metarhodopsin I/metarhodopsin II equilibrium of bovine rhodopsin by ionic strength--evidence for a surface-charge effect. Eur J Biochem. 1997 Jan 15;243(1-2):174–180. doi: 10.1111/j.1432-1033.1997.0174a.x. [DOI] [PubMed] [Google Scholar]
  6. Fahmy K., Sakmar T. P. Regulation of the rhodopsin-transducin interaction by a highly conserved carboxylic acid group. Biochemistry. 1993 Jul 20;32(28):7229–7236. doi: 10.1021/bi00079a020. [DOI] [PubMed] [Google Scholar]
  7. Ferreira P. A., Nakayama T. A., Travis G. H. Interconversion of red opsin isoforms by the cyclophilin-related chaperone protein Ran-binding protein 2. Proc Natl Acad Sci U S A. 1997 Feb 18;94(4):1556–1561. doi: 10.1073/pnas.94.4.1556. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ganter U. M., Schmid E. D., Siebert F. The photoreaction of vacuum-dried rhodopsin at low temperature: evidence for charge stabilization by water. J Photochem Photobiol B. 1988 Dec;2(4):417–426. doi: 10.1016/1011-1344(88)85070-x. [DOI] [PubMed] [Google Scholar]
  9. Imai H., Imamoto Y., Yoshizawa T., Shichida Y. Difference in molecular properties between chicken green and rhodopsin as related to the functional difference between cone and rod photoreceptor cells. Biochemistry. 1995 Aug 22;34(33):10525–10531. doi: 10.1021/bi00033a026. [DOI] [PubMed] [Google Scholar]
  10. Imai H., Kojima D., Oura T., Tachibanaki S., Terakita A., Shichida Y. Single amino acid residue as a functional determinant of rod and cone visual pigments. Proc Natl Acad Sci U S A. 1997 Mar 18;94(6):2322–2326. doi: 10.1073/pnas.94.6.2322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Janssen J. J., Bovee-Geurts P. H., Merkx M., DeGrip W. J. Histidine tagging both allows convenient single-step purification of bovine rhodopsin and exerts ionic strength-dependent effects on its photochemistry. J Biol Chem. 1995 May 12;270(19):11222–11229. doi: 10.1074/jbc.270.19.11222. [DOI] [PubMed] [Google Scholar]
  12. Kazmi M. A., Dubin R. A., Oddoux C., Ostrer H. High-level inducible expression of visual pigments in transfected cells. Biotechniques. 1996 Aug;21(2):304–311. doi: 10.2144/96212rr05. [DOI] [PubMed] [Google Scholar]
  13. Kazmi M. A., Sakmar T. P., Ostrer H. Mutation of a conserved cysteine in the X-linked cone opsins causes color vision deficiencies by disrupting protein folding and stability. Invest Ophthalmol Vis Sci. 1997 May;38(6):1074–1081. [PubMed] [Google Scholar]
  14. Kojima D., Imai H., Okano T., Fukada Y., Crescitelli F., Yoshizawa T., Shichida Y. Purification and low temperature spectroscopy of gecko visual pigments green and blue. Biochemistry. 1995 Jan 24;34(3):1096–1106. doi: 10.1021/bi00003a047. [DOI] [PubMed] [Google Scholar]
  15. Kojima D., Oura T., Hisatomi O., Tokunaga F., Fukada Y., Yoshizawa T., Shichida Y. Molecular properties of chimerical mutants of gecko blue and bovine rhodopsin. Biochemistry. 1996 Feb 27;35(8):2625–2629. doi: 10.1021/bi9511548. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lewis J. W., van Kuijk F. J., Carruthers J. A., Kliger D. S. Metarhodopsin III formation and decay kinetics: comparison of bovine and human rhodopsin. Vision Res. 1997 Jan;37(1):1–8. doi: 10.1016/s0042-6989(96)00138-1. [DOI] [PubMed] [Google Scholar]
  18. Liang J., Govindjee R., Ebrey T. G. Metarhodopsin intermediates of the gecko cone pigment P521. Biochemistry. 1993 Dec 28;32(51):14187–14193. doi: 10.1021/bi00214a018. [DOI] [PubMed] [Google Scholar]
  19. Merbs S. L., Nathans J. Absorption spectra of human cone pigments. Nature. 1992 Apr 2;356(6368):433–435. doi: 10.1038/356433a0. [DOI] [PubMed] [Google Scholar]
  20. Merbs S. L., Nathans J. Absorption spectra of the hybrid pigments responsible for anomalous color vision. Science. 1992 Oct 16;258(5081):464–466. doi: 10.1126/science.1411542. [DOI] [PubMed] [Google Scholar]
  21. Nathans J., Thomas D., Hogness D. S. Molecular genetics of human color vision: the genes encoding blue, green, and red pigments. Science. 1986 Apr 11;232(4747):193–202. doi: 10.1126/science.2937147. [DOI] [PubMed] [Google Scholar]
  22. Nishimura S., Sasaki J., Kandori H., Lugtenburg J., Maeda A. Structural changes in the lumirhodopsin-to-metarhodopsin I conversion of air-dried bovine rhodopsin. Biochemistry. 1995 Dec 26;34(51):16758–16763. doi: 10.1021/bi00051a025. [DOI] [PubMed] [Google Scholar]
  23. Okada T., Matsuda T., Kandori H., Fukada Y., Yoshizawa T., Shichida Y. Circular dichroism of metaiodopsin II and its binding to transducin: a comparative study between meta II intermediates of iodopsin and rhodopsin. Biochemistry. 1994 Apr 26;33(16):4940–4946. doi: 10.1021/bi00182a024. [DOI] [PubMed] [Google Scholar]
  24. Okano T., Fukada Y., Artamonov I. D., Yoshizawa T. Purification of cone visual pigments from chicken retina. Biochemistry. 1989 Oct 31;28(22):8848–8856. doi: 10.1021/bi00448a025. [DOI] [PubMed] [Google Scholar]
  25. Okano T., Kojima D., Fukada Y., Shichida Y., Yoshizawa T. Primary structures of chicken cone visual pigments: vertebrate rhodopsins have evolved out of cone visual pigments. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5932–5936. doi: 10.1073/pnas.89.13.5932. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Oprian D. D., Asenjo A. B., Lee N., Pelletier S. L. Design, chemical synthesis, and expression of genes for the three human color vision pigments. Biochemistry. 1991 Dec 3;30(48):11367–11372. doi: 10.1021/bi00112a002. [DOI] [PubMed] [Google Scholar]
  27. Pace C. N., Vajdos F., Fee L., Grimsley G., Gray T. How to measure and predict the molar absorption coefficient of a protein. Protein Sci. 1995 Nov;4(11):2411–2423. doi: 10.1002/pro.5560041120. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Phillips W. J., Cerione R. A. The intrinsic fluorescence of the alpha subunit of transducin. Measurement of receptor-dependent guanine nucleotide exchange. J Biol Chem. 1988 Oct 25;263(30):15498–15505. [PubMed] [Google Scholar]
  29. Rafferty C. N., Shichi H. The involvement of water at the retinal binding site in rhodopsin and early light-induced intramolecular proton transfer. Photochem Photobiol. 1981 Feb;33(2):229–234. doi: 10.1111/j.1751-1097.1981.tb05329.x. [DOI] [PubMed] [Google Scholar]
  30. Schnapf J. L., Kraft T. W., Baylor D. A. Spectral sensitivity of human cone photoreceptors. 1987 Jan 29-Feb 4Nature. 325(6103):439–441. doi: 10.1038/325439a0. [DOI] [PubMed] [Google Scholar]
  31. Shichida Y., Imai H., Imamoto Y., Fukada Y., Yoshizawa T. Is chicken green-sensitive cone visual pigment a rhodopsin-like pigment? A comparative study of the molecular properties between chicken green and rhodopsin. Biochemistry. 1994 Aug 9;33(31):9040–9044. doi: 10.1021/bi00197a002. [DOI] [PubMed] [Google Scholar]
  32. Shichida Y., Okada T., Kandori H., Fukada Y., Yoshizawa T. Nanosecond laser photolysis of iodopsin, a chicken red-sensitive cone visual pigment. Biochemistry. 1993 Oct 12;32(40):10832–10838. doi: 10.1021/bi00091a039. [DOI] [PubMed] [Google Scholar]
  33. Vissers P. M., DeGrip W. J. Functional expression of human cone pigments using recombinant baculovirus: compatibility with histidine tagging and evidence for N-glycosylation. FEBS Lett. 1996 Oct 28;396(1):26–30. doi: 10.1016/0014-5793(96)01064-2. [DOI] [PubMed] [Google Scholar]
  34. Wang Z., Asenjo A. B., Oprian D. D. Identification of the Cl(-)-binding site in the human red and green color vision pigments. Biochemistry. 1993 Mar 9;32(9):2125–2130. doi: 10.1021/bi00060a001. [DOI] [PubMed] [Google Scholar]
  35. Yoshizawa T., Wald G. Photochemistry of lodopsin. Nature. 1967 May 6;214(5088):566–571. doi: 10.1038/214566a0. [DOI] [PubMed] [Google Scholar]
  36. van Breugel P. J., Geurts P. H., Daemen F. J., Bonting S. L. Biochemical aspects of the visual process. XXXVIII. Effects of lateral aggregation on rhodopsin in phospholipase C-treated photoreceptor membranes. Biochim Biophys Acta. 1978 May 4;509(1):136–147. doi: 10.1016/0005-2736(78)90014-7. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES