Assays of retinoid dehydrogenases by phase partition.
Two modifications of an extraction assay for retinoid dehydrogenases are described. The first method involves the transfer of tritium from carbon-15 of [15-3H]-retinol or [15-3H]retinaldehyde to NAD, whereas in the second method, tritium from [3H]NADPH is transferred to all-trans-retinaldehyde. Since both versions of the assay involve the interconversion of water-soluble and -insoluble tritium-labeled compounds, a simple phase partition is sufficient to separate labeled products from labeled reactants. The assays are shown to provide reliable estimations of the reaction progress for three retinoid dehydrogenases of the visual system when compared to HPLC analysis of retinoid products or gel filtration analysis of pyridine nucleotide. The assays will be useful in studying retinoid dehydrogenases from other tissues and in principle can be modified for other dehydrogenase reactions with water-insoluble substrates.
Saari
J C
JC
Department of Ophthalmology, University of Washington School of Medicine, Seattle 98195.
Bredberg
D L
DL
Garwin
G G
GG
Buczyłko
J
J
Wheeler
T
T
Palczewski
K
K
D000429:Alcohol Oxidoreductases / Q000032:analysis* / Q000302:isolation & purification; D000444:Aldehyde Dehydrogenase / Q000032:analysis* / Q000302:isolation & purification; D000818:Animals; D002417:Cattle; D002850:Chromatography, Gel / Q000379:methods; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007700:Kinetics; D009249:NADP / Q000378:metabolism; D015203:Reproducibility of Results; D012160:Retina / Q000201:enzymology*; D017949:Retinal Cone Photoreceptor Cells / Q000201:enzymology; D012172:Retinaldehyde / Q000378:metabolism; D014316:Tritium; D014801:Vitamin A / Q000378:metabolism
D014316:Tritium; D014801:Vitamin A; D009249:NADP; D000429:Alcohol Oxidoreductases; C022520:retinol dehydrogenase; D000444:Aldehyde Dehydrogenase; D012172:Retinaldehyde