Molecular Biology and Analytical Chemistry Methods Used to Probe the Retinoid Cycle
Open accessAbstract
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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UV/Vis detection offers retinoid quantification at low picomolar range through most photodiode array detectors and linearity over 2-1500 pmol.
“UV/Vis detection offers a limit of retinoid quantification at a low picomolar range through most photodiode array detectors and provides excellent linearity over a wide range of concentrations (2–1500 pmol) (Figure 6C).163”
From Chemistry of the Retinoid (Visual) Cycle (2014) · cites this paper as [163]
The Introduction reports quantification down to approximately 2 pmol by HPLC with photodiode-array detection and excellent standard-curve linearity over 2–1500 pmol. It does not establish that this applies to most photodiode-array detectors, and pmol (an amount) is not the same as a picomolar concentration. (LLM, conf=0.86)
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Chemistry of the Retinoid (Visual) Cycle
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Self-citation
- Authors
- Golczak, M., Bereta, G., Maeda, A., & Palczewski, K.
- Affiliations
- Case Western Reserve University (US)
- Venue
- Methods in Molecular Biology
- Publisher
- Humana Press
- Published
- 2010
- Volume
- 652
- Pages
- 229-245
- ISSN
- 1064-3745 (ISSN-L)
- 1940-6029
- PMC
- PMC4607022
- OpenAlex
- W2428471783
- Cited by
- 16
- References
- 31
- Citations by year
- 2025: 1 · 2024: 1 · 2022: 1 · 2020: 1 · 2019: 1 · 2018: 1 · 2017: 3 · 2016: 2 · 2015: 2 · 2013: 1
- FWCI
- 1.16
- Type
- book chapter
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- Methods in molecular biology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Funders
- National Eye Institute (K08EY019880, EY009339, K08EY019031, P30 EY-11373, P30EY011373, R01EY009339)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinal Development and Disorders
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Retinoids in leukemia and cellular processes
- Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Photoreceptor and optogenetics research
- Keywords
- retinoid cycle, 11-cis-retinal regeneration, rhodopsin, RPE65, vitamin A metabolism, retinoid homeostasis, retinoid derivatives, protein stability, enzyme activity, congenital blindness, mass spectrometry, HPLC
- MeSH terms
- Retinoid Isomerohydrolase, Acyltransferases, Animals, Carrier Proteins, Chemistry Techniques, Analytical, Chromatography, High Pressure Liquid, Chromatography, Liquid, Esters, Eye, Eye Proteins, Isomerism, Lipofuscin, Molecular Biology, Pyridinium Compounds, Retinaldehyde, Retinoids, Mass Spectrometry, Vitamin A, cis-trans-Isomerases, NIH 3T3 Cells, Mice
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