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O. Nikolaeva, G. Moiseyev, J.-X. Ma; RPE65 - Phospholipid Membrane Binding is Enhanced by the Presence of Retinyl Ester and Causes RPE65 Conformation Changes. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4811.
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RPE65 is a key enzyme of the visual cycle, converting retinyl ester to 11-cis retinol. It has been shown that the association of RPE65 with the membrane is required for its isomerohydrolase activity.The purpose of this study is to evaluate the impact of the presence of retinyl ester on RPE65’s binding affinity to the phospholipid membrane as well as the conformational change of RPE65 induced by binding with the membrane.
Recombinant chicken RPE65 was expressed in 293-LRAT cells using an adenovirus vector and purified to homogeneity. The flotations in a sucrose gradient and fluorescent spectroscopy were used to evaluate RPE65-membrane binding affinity. Circular dichroism (CD) was applied to observe RPE65 conformation changes. RPE65 isomerohydrolase activity was analyzed by HPLC using retinyl palmitate incorporated into liposomes.
Our fluorescence and flotation results have shown that the binding affinity of RPE65 to the phospholipid membrane (liposomes) was increased by the presence of retinyl palmitate in the membrane. The increase in affinity was more pronounced for the membranes composed of positively charged phospholipids. Binding of RPE65 with different phospholipid membranes induced different degrees of conformational change of RPE65, which were correlated with its isomerohydrolase activity.
The presence of retinyl palmitate in the phospholipid membrane improves RPE65 membrane binding. Isomerohydrolase activity of RPE65 depends on the content of the phospholipid membrane and the conformational changes of RPE65 induced by membrane binding.
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