Molecular biology and analytical chemistry methods used to probe the retinoid cycle.
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.
Golczak
Marcin
M
Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, OH 44106-4965, USA.
Bereta
Grzegorz
G
Maeda
Akiko
A
Palczewski
Krzysztof
K
D000217:Acyltransferases / Q000378:metabolism; D000818:Animals; D002352:Carrier Proteins / Q000378:metabolism; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D004952:Esters; D005123:Eye / Q000378:metabolism; D005136:Eye Proteins / Q000378:metabolism; D007536:Isomerism; D008062:Lipofuscin / Q000378:metabolism; D013058:Mass Spectrometry; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D041681:NIH 3T3 Cells; D011726:Pyridinium Compounds / Q000378:metabolism; D012172:Retinaldehyde / Q000031:analogs & derivatives / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012176:Retinoids / Q000032:analysis / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D014801:Vitamin A / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D000217:Acyltransferases; D002352:Carrier Proteins; D004952:Esters; D005136:Eye Proteins; D008062:Lipofuscin; D011726:Pyridinium Compounds; D012172:Retinaldehyde; D012176:Retinoids; D014801:Vitamin A; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C112040:A2-E (N-retinylidene-N-retinylethanolamine); C525876:retinal dimer; C056942:lecithin-retinol acyltransferase