April 1995
Volume 36, Issue 5
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Articles  |   April 1995
Rhodopsin from the fish, Astyanax: role of tyrosine 261 in the red shift.
Author Affiliations
  • R Yokoyama
    Department of Biology, Syracuse University, NY 13244, USA.
  • B E Knox
    Department of Biology, Syracuse University, NY 13244, USA.
  • S Yokoyama
    Department of Biology, Syracuse University, NY 13244, USA.
Investigative Ophthalmology & Visual Science April 1995, Vol.36, 939-945. doi:
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    • Get Citation

      R Yokoyama, B E Knox, S Yokoyama; Rhodopsin from the fish, Astyanax: role of tyrosine 261 in the red shift.. Invest. Ophthalmol. Vis. Sci. 1995;36(5):939-945.

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Abstract

PURPOSE: To isolate and characterize the rhodopsin cDNA from the fish, Astyanax fasciatus, and to determine the effect of tyrosine 261 on its spectral tuning. METHODS: The rhodopsin cDNA was cloned using reverse transcription-polymerase chain reaction amplification and then sequenced. A mutant, Y261F, was generated by site-directed mutagenesis. Both wild type and mutant were transiently expressed in COS-1 cells, regenerated with 11-cis retinal, and purified by immunoaffinity chromatography. Ultraviolet-visible spectrophotometry was used to determine wavelength of maximum absorption. RESULTS: A fasciatus rhodopsin cDNA exhibits 80% amino acid identity with bovine rhodopsin. In contrast to all known rhodopsins, this rhodopsin contains a tyrosine instead of a phenylalanine at amino acid position 261. Indeed, this particular amino acid replacement has been implicated in the long wavelength absorption of the red cone pigment. Site-directed mutagenesis was used to change the Astyanax amino acid 261 to phenylalanine (Y261F). Expression of the Y261F mutant in COS-1 cells showed an absorbance maximum of 496 nm, compared to 504 nm for the wild type pigment. CONCLUSIONS: A naturally occurring fish rhodopsin is red shifted about 8 nm due to one critical amino acid substitution.

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