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Real-time Analyses of Retinol Transport by the Membrane Receptor of Plasma Retinol Binding Protein
Authors:Riki Kawaguchi  Ming Zhong  Hui Sun
Institution:Department of Physiology, Jules Stein Eye Institute and Howard Hughes Medical Institute, University of California, Los Angeles
Abstract:Vitamin A is essential for vision and the growth/differentiation of almost all human organs. Plasma retinol binding protein (RBP) is the principle and specific carrier of vitamin A in the blood. Here we describe an optimized technique to produce and purify holo-RBP and two real-time monitoring techniques to study the transport of vitamin A by the high-affinity RBP receptor STRA6. The first technique makes it possible to produce a large quantity of high quality holo-RBP (100%-loaded with retinol) for vitamin A transport assays. High quality RBP is essential for functional assays because misfolded RBP releases vitamin A readily and bacterial contamination in RBP preparation can cause artifacts. Real-time monitoring techniques like electrophysiology have made critical contributions to the studies of membrane transport. The RBP receptor-mediated retinol transport has not been analyzed in real time until recently. The second technique described here is the real-time analysis of STRA6-catalyzed retinol release or loading. The third technique is real-time analysis of STRA6-catalyzed retinol transport from holo-RBP to cellular retinol binding protein I (CRBP-I). These techniques provide high sensitivity and resolution in revealing RBP receptor''s vitamin A uptake mechanism.
Keywords:Biochemistry  Issue 71  Molecular Biology  Genetics  Cellular Biology  Molecular Biology  Anatomy  Physiology  Ophthalmology  Proteomics  Proteins  Membrane Transport Proteins  Vitamin A  retinoid  RBP complex  membrane transport  membrane receptor  STRA6  retinol binding protein
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