Light-dependent activation of transducin by pineal opsin

M. Max, A. Surya, J. S. Takahashi, R. F. Margolskee, B. E. Knox

Research output: Contribution to journalArticle

Abstract

Purpose: To express the chick pineal opsin, P-opsin, in a pineal cell line, purify the photopigment and characterize its interaction with rod transducin. Methods. A P-opsin cDNA, containing an epitope for a monoclonal antibody to bovine rhodopsin, was expressed in the CP3 cell line. Photopigment was generated by incubation with 11-cis retinal, solubilized in dodecyl maltoside and purified by immunoaffinity chromatography. UV/Vis spectra were measured and activation of purified bovine transducin (GT) was assayed using a [35S]GTPγS filter binding assay. Results: The UV/Vis spectrum of P-opsin had a maximum absorbance at 465 nm, which bleached to 380 nm when exposed to light. In the presence of 50 mM NH2OH in the dark, P-opsin slowly bleached (half-time of ∼2 h). Light-exposed P-opsin stimulated guanyl nucleotide exchange on bovine rod GT. In exhaustive binding assays, up to one mol of GT was activated per mol of P-opsin, similar to that observed with purified cone opsins, but ∼100-fold less than with bovine rhodopsin The kinetics of P-opsin was comparable to rhodopsin. Half-maximal activation occured with ion 1.5 min. However, total turnovers were less for P-opsin: 0.8 mol GT/mol P-opsin/min compared to 105 mol Gr/mol rhodopsin/min. The lifetime of the active species formed following photobleaching of P-opsin was 6 min, shorter than the 30 min found for rhodopsin. Conclusion: Pineal opsin is a photopigment that binds 11-cis retinal and activates transducin in a light dependent manner. P-opsin has spectral and biochemical characteristics that are intermediate between rod and cone opsins. These results indicate a role for P-opsin in pineal phototransduction and circadian phase-shifting.

Original languageEnglish (US)
JournalInvestigative Ophthalmology and Visual Science
Volume37
Issue number3
StatePublished - Feb 15 1996

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Transducin
Opsins
Light
Rhodopsin
Cone Opsins
Retinaldehyde
Rod Opsins
Light Signal Transduction
Photobleaching
Cell Line
Vertebrate Photoreceptor Cells

ASJC Scopus subject areas

  • Ophthalmology

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Light-dependent activation of transducin by pineal opsin. / Max, M.; Surya, A.; Takahashi, J. S.; Margolskee, R. F.; Knox, B. E.

In: Investigative Ophthalmology and Visual Science, Vol. 37, No. 3, 15.02.1996.

Research output: Contribution to journalArticle

Max, M. ; Surya, A. ; Takahashi, J. S. ; Margolskee, R. F. ; Knox, B. E. / Light-dependent activation of transducin by pineal opsin. In: Investigative Ophthalmology and Visual Science. 1996 ; Vol. 37, No. 3.
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abstract = "Purpose: To express the chick pineal opsin, P-opsin, in a pineal cell line, purify the photopigment and characterize its interaction with rod transducin. Methods. A P-opsin cDNA, containing an epitope for a monoclonal antibody to bovine rhodopsin, was expressed in the CP3 cell line. Photopigment was generated by incubation with 11-cis retinal, solubilized in dodecyl maltoside and purified by immunoaffinity chromatography. UV/Vis spectra were measured and activation of purified bovine transducin (GT) was assayed using a [35S]GTPγS filter binding assay. Results: The UV/Vis spectrum of P-opsin had a maximum absorbance at 465 nm, which bleached to 380 nm when exposed to light. In the presence of 50 mM NH2OH in the dark, P-opsin slowly bleached (half-time of ∼2 h). Light-exposed P-opsin stimulated guanyl nucleotide exchange on bovine rod GT. In exhaustive binding assays, up to one mol of GT was activated per mol of P-opsin, similar to that observed with purified cone opsins, but ∼100-fold less than with bovine rhodopsin The kinetics of P-opsin was comparable to rhodopsin. Half-maximal activation occured with ion 1.5 min. However, total turnovers were less for P-opsin: 0.8 mol GT/mol P-opsin/min compared to 105 mol Gr/mol rhodopsin/min. The lifetime of the active species formed following photobleaching of P-opsin was 6 min, shorter than the 30 min found for rhodopsin. Conclusion: Pineal opsin is a photopigment that binds 11-cis retinal and activates transducin in a light dependent manner. P-opsin has spectral and biochemical characteristics that are intermediate between rod and cone opsins. These results indicate a role for P-opsin in pineal phototransduction and circadian phase-shifting.",
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