TY - JOUR
T1 - [12] Purification of Interstitial Retinol-Binding Protein from the Eye
AU - Fong, S. L.
AU - Liou, G. I.
AU - Bridges, C. D.B.
PY - 1986/1/1
Y1 - 1986/1/1
N2 - This chapter describes the purification of interstitial retinol-binding protein (IRBP) from the eye. IRBP is synthesized and secreted by cells of the neural retina, which may therefore have an important function in regulating its amount in the subretinal space. Purified bovine IRBP binds approximately two molecules of all-trans-retinol and contains 8.4% by weight of carbohydrate that consists of sialic acid, neutral hexoses (mannose, fucose, galactose), and glucosamine in the molar ratio of approximately 1:3:2. The purification of IRBP to homogeneity by high-performance size-exclusion chromatography of crude bovine interphotoreceptor matrix (IPM) is described. Sufficient IRBP is obtained by this method to demonstrate that it consisted of a single concanavalin A (Con A)-binding polypeptide with apparent Mr of 140–145K by SDS–polyacrylamide gel electrophoresis. The anion-exchange chromatography on DEAE-cellulose (DE-52) followed by gel-filtration chromatography on Sepharose CL-4B can be used to obtain purified preparations of bovine and human IRBPs so permitting their partial characterization. For rat IRBP used, Con A-Sepharose can be followed by anion-exchange chromatography on DEAE-cellulose. The preparation of bovine IRBP in milligram quantities is described in the chapter.
AB - This chapter describes the purification of interstitial retinol-binding protein (IRBP) from the eye. IRBP is synthesized and secreted by cells of the neural retina, which may therefore have an important function in regulating its amount in the subretinal space. Purified bovine IRBP binds approximately two molecules of all-trans-retinol and contains 8.4% by weight of carbohydrate that consists of sialic acid, neutral hexoses (mannose, fucose, galactose), and glucosamine in the molar ratio of approximately 1:3:2. The purification of IRBP to homogeneity by high-performance size-exclusion chromatography of crude bovine interphotoreceptor matrix (IPM) is described. Sufficient IRBP is obtained by this method to demonstrate that it consisted of a single concanavalin A (Con A)-binding polypeptide with apparent Mr of 140–145K by SDS–polyacrylamide gel electrophoresis. The anion-exchange chromatography on DEAE-cellulose (DE-52) followed by gel-filtration chromatography on Sepharose CL-4B can be used to obtain purified preparations of bovine and human IRBPs so permitting their partial characterization. For rat IRBP used, Con A-Sepharose can be followed by anion-exchange chromatography on DEAE-cellulose. The preparation of bovine IRBP in milligram quantities is described in the chapter.
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U2 - 10.1016/S0076-6879(86)23014-1
DO - 10.1016/S0076-6879(86)23014-1
M3 - Article
C2 - 3702708
AN - SCOPUS:0022558982
SN - 0076-6879
VL - 123
SP - 102
EP - 111
JO - Methods in Enzymology
JF - Methods in Enzymology
IS - C
ER -