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 genesencoding proteins involved in the retinoid cycle lead to abnormalities in retinoid homeostasis and numerous congenital blindingdiseases of humans. Thus, elucidation of disease-specific changes in enzymatic activities and retinoid content of the retinacan provide important insights into the mechanisms of disease initiation and progression. Here, we use the protein RPE65 asan example to describe generally applicable methods for determining the stability and enzymatic activity of proteins and theirmutants involved in retinoid metabolism. Additionally, we introduce a range of analytical techniques involving high-performanceliquid chromatography and mass spectrometry to detect and quantify retinoids and their derivatives in eye extracts. Biochemicalprotocols combined with advanced mass spectrometry should facilitate fundamental biological studies of vision.