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Retinoids pp 229-245 | Cite as

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

  • Marcin Golczak
  • Grzegorz Bereta
  • Akiko Maeda
  • Krzysztof Palczewski
Protocol
Part of the Methods in Molecular Biology book series (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.

Key words

RPE65 retinoid isomerization mass spectrometry A2E retinal dimer 

Notes

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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Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Marcin Golczak
    • 1
  • Grzegorz Bereta
    • 1
  • Akiko Maeda
    • 1
  • Krzysztof Palczewski
    • 1
  1. 1.Department of Pharmacology, School of MedicineCase Western Reserve UniversityClevelandUSA

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