June 2013
Volume 54, Issue 15
Free
ARVO Annual Meeting Abstract  |   June 2013
Substrate Specificity and Localization of AL-OL Coupling Reaction in Carp Cones
Author Affiliations & Notes
  • Satoru Kawamura
    Grad Sch of Frontier Biosci, Osaka Univ, Suita, Japan
    Dept of Biol Sci, Faculty of Sci, Osaka Univ, Toyonaka, Japan
  • Shinya Sato
    Dept of Biol Sci, Faculty of Sci, Osaka Univ, Toyonaka, Japan
  • Shuji Tachibanaki
    Grad Sch of Frontier Biosci, Osaka Univ, Suita, Japan
    Dept of Biol Sci, Faculty of Sci, Osaka Univ, Toyonaka, Japan
  • Takashi Fukagawa
    Grad Sch of Frontier Biosci, Osaka Univ, Suita, Japan
  • Footnotes
    Commercial Relationships Satoru Kawamura, None; Shinya Sato, None; Shuji Tachibanaki, None; Takashi Fukagawa, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2013, Vol.54, 1701. doi:
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      Satoru Kawamura, Shinya Sato, Shuji Tachibanaki, Takashi Fukagawa; Substrate Specificity and Localization of AL-OL Coupling Reaction in Carp Cones. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1701.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Cones are known to regenerate visual pigments from 11-cis retinol. Our previous study showed that carp cones specifically have an enzyme activity to oxidize 11-cis retinol (alcohol) to 11-cis retinal (aldehyde) with concomitant reduction of all-trans retinal to all-trans retinol (AL-OL coupling reaction, Miyazono et al., 2008, PNAS 105: 16051). In this reaction, it was not necessary to add NADP+, a cofactor required for a conventional retinol dehydrogenase activity. In the present study, we tried to examine the substrate specificity and localization of this AL-OL coupling reaction.

Methods: Cones were isolated from carp retinas using a stepwise Percoll density gradient (Tachibanaki et al., PNAS, 102: 9329). Substrate specificity was examined by a pair of alcohol and aldehyde of various retinoids and related compounds. Reaction products were analyzed and quantified with HPLC. To examine the localization of the reaction, the activity was first measured in the membrane fraction and in the soluble fraction of our cone preparation, and then in a cone outer segment (COS)-rich and cone inner segment (CIS)-rich membrane fractions prepared by centrifugation after mechanical treatment of purified cones.

Results: In the presence of all-trans retinal, 11-cis retinol and 9-cis retinol were oxidized, but other alcohols including all-trans retinol and 13-cis retinol were not oxidized. Besides all-trans retinal, hydrophobic aldehydes such as benzaldehyde and dodecanal were effective in the oxidation of 11-cis retinol. AL-OL coupling activity was found in the membrane fraction, and was not co-localized with visual pigment. The activity was extracted by a detergent.

Conclusions: Substrate specificity for oxidation of alcohol was high, but that for reduction of aldehyde was low. Presumably, the binding site for alcohol is specific only for 11-cis and 9-cis form of retinol, but the site for aldehyde accepts a wide range of hydrophobic aldehydes. This AL-OL coupling activity seems to be localized in the CIS membrane. These findings suggest that in the AL-OL coupling reaction, not only all-trans retinal produced by visual pigment bleach but also other hydrophobic aldehydes produced in the CIS could be the substrate to oxidize 11-cis retinol.

Keywords: 648 photoreceptors • 705 retinoids/retinoid binding proteins • 514 enzymes/enzyme inhibitors  
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