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Molecular Biology and Analytical Chemistry Methods Used to Probe the Retinoid Cycle

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Retinoids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 652))

Abstract

The retinoid (visual) cycle is a complex enzymatic pathway essential for regeneration of the visual chromophore, 11-cis-retinal, a component of rhodopsin that undergoes activation by light in vertebrate eyes. Pathogenic mutations within genes encoding proteins involved in the retinoid cycle lead to abnormalities in retinoid homeostasis and numerous congenital blinding diseases of humans. Thus, elucidation of disease-specific changes in enzymatic activities and retinoid content of the retina can provide important insights into the mechanisms of disease initiation and progression. Here, we use the protein RPE65 as an example to describe generally applicable methods for determining the stability and enzymatic activity of proteins and their mutants involved in retinoid metabolism. Additionally, we introduce a range of analytical techniques involving high-performance liquid chromatography and mass spectrometry to detect and quantify retinoids and their derivatives in eye extracts. Biochemical protocols combined with advanced mass spectrometry should facilitate fundamental biological studies of vision.

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Acknowledgment

This research was supported in part by grants EY009339 and P30 EY11373 from the National Institutes of Health and the Foundation Fighting Blindness.

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Authors and Affiliations

  1. Department of Pharmacology, School of Medicine, Case Western Reserve University, 10900 Euclid Ave, Cleveland, OH, 44106-4965, USA

    Marcin Golczak, Grzegorz Bereta, Akiko Maeda & Krzysztof Palczewski

Authors
  1. Marcin Golczak
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  2. Grzegorz Bereta
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  3. Akiko Maeda
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  4. Krzysztof Palczewski
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Editors and Affiliations

  1. David Geffen School of Medicine, Dept. Physiology, University of California, Los Angeles, Charles Young Dr. S. 650, Los Angeles, 90095-1751, California, USA

    Hui Sun

  2. , Jules Stein Eye Institute, UCLA School of Medicine, 100 Stein Plaza, BH-667, Los Angeles, 90095, California, USA

    Gabriel H. Travis

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Golczak, M., Bereta, G., Maeda, A., Palczewski, K. (2010). Molecular Biology and Analytical Chemistry Methods Used to Probe the Retinoid Cycle. In: Sun, H., Travis, G. (eds) Retinoids. Methods in Molecular Biology, vol 652. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-325-1_13

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  • DOI: https://doi.org/10.1007/978-1-60327-325-1_13

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-324-4

  • Online ISBN: 978-1-60327-325-1

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