ABSTRACT
The exchange of all-trans retinoids (retinal, retinol, retinylpalmitate) between PC-vesicles, PC-vesicles and liver microsomes or PC-vesicles and rod outer segment membranes is investigated using 11,12(3)H labeled compounds. In the first two systems, retinal and retinol exchange rapidly, retinyl acetate slowly and retinyl palmitate not at all. Rod outer segment membranes however take up relatively small amounts of retinoids (retinylpalmitate less than retinol less than retinal) and rapidly lose 60-90% of their label in the presence of PC-vesicles. E.G. retinoids clearly favour the PC-vesicle membrane. Apparently, rod outer segment membranes have a much lower affinity for retinoids than other artificial or natural membranes investigated so far.
Subject(s)
Photoreceptor Cells/metabolism , Retinoids/metabolism , Rod Cell Outer Segment/metabolism , Animals , Cattle , Diterpenes , Liposomes , Membranes/metabolism , Microsomes, Liver/metabolism , Retinaldehyde/metabolism , Retinyl Esters , Vitamin A/analogs & derivatives , Vitamin A/metabolismABSTRACT
1. Dark incubation of retinoids (retinyl ester, retinol, retinal, retinaloxime) in suspensions of rod outer segment membranes leads to substantial isomerization (and partial degradation) in the case of retinals only. 2. All-trans, 13-cis and 9-cis-retinal all isomerize at the delta 13 double bond leading to an equilibrium with approximately 75% trans and 25% cis isomer at this bond (all-trans in equilibrium 13-cis and 9-cis in equilibrium 9,13-dicis). 11-cis-Retinal isomerizes irreversibly to a mixture of all-trans and 13-cis-retinal. 3. The active compound appears to be phosphatidylethanolamine present in the membrane. The amino group and the phosphate, as well as the hydrophobic part of the phospholipid are essential. 4. At least three factors are important for the phosphatidylethanolamine-catalyzed isomerization as studied with the 13-cis isomer: the concentration of phosphatidylethanolamine, the concentration of Schiff base between retinal and phosphatidylethanolamine and the presence of lipid aggregates. 5. Based on these observations a mechanism is proposed, which satisfactorily explains the specificity of the isomerization pattern. 6. It is suggested that reisomerization of all-trans to 11-cis retinal in vivo takes place by fixation of all-trans retinal on an adequate surface (e.g. opsin) and a localized nucleophilic attack on the C-11 atom, followed by trapping of the isomerized chromophore by opsin. 7. It is further concluded that retinal does not occur in vivo as a free intermediate. Direct transfer from one protein to another (opsin, retinol dehydrogenase, retinal binding proteins) seems to take place.
Subject(s)
Phosphatidylethanolamines , Retinaldehyde/analogs & derivatives , Vitamin A/analogs & derivatives , Catalysis , Chemical Phenomena , Chemistry , Darkness , In Vitro Techniques , Models, Chemical , Models, Molecular , Rod Cell Outer Segment , StereoisomerismABSTRACT
A method is described for the quantitative extraction of retinal in its original isomeric configuration from retinal-containing pigments. Using excess of hydroxylamine under denaturing conditions, the chromophore of retinal bearing natural products is converted into the corresponding retinaloxime with complete retention of geometric configuration. The retinaloximes can be quantitatively extracted with dichloromethane and analyzed by high-performance liquid chromatography.
Subject(s)
Retinaldehyde/analysis , Vitamin A/analogs & derivatives , Animals , Chromatography, High Pressure Liquid , Hydroxylamines , Light , Photoreceptor Cells/analysis , Rhodopsin/analysis , StereoisomerismABSTRACT
A method for the analytical and/or preparative separation of the 13-cis, 11-cis, 9-cis and all all-trans isomers of retinyl palmitate ester, retinal and retinol by high-performance liquid chromatography is described. A straight-phase adsorption system consisting of Si 60 silica gel as the stationary phase and mixtures of n-hexane and dioxane as mobile phases were employed, using photometric detection at either 320 or 360 nm, depending on the nature of the compounds being studied. With simple adaptation of the phase system, all geometric isomers within each group could be separated. The precision of the method was 0.5% at the 10-microgram (about 40-nmole) level and 4% at the 10-ng (about 40-pmole) level (n = 3). The detection limit was about 0.5 ng (about 2 pmole). The suitability of the phase system is demonstrated by chromatograms of test mixtures and of eye extracts. The fractionation of 0.5 mg of an isomeric sample could be achieved on a column of length 250 mm and I.D. 10 mm.
