Solubilization and purification of the retinol-binding protein receptor from human placental membranes.
The membrane receptor for retinol-binding protein (RBP) has been solubilized from human placental brush-border membranes with octyl-beta-glucoside, Nonidet P-40 and CHAPS. A method, based on the preferential precipitation of 125I-RBP-receptor complex with poly(ethylene glycol) 8000, was developed in order to measure the RBP-binding activity in the detergent extracts. The receptor was fairly stable (4 degrees C, 7 days) in octyl-beta-glucoside and Nonidet P-40, but quickly lost activity in CHAPS. The detergent-solubilized form retained all the properties characteristic of the membrane-bound protein, except for a small decrease in affinity for RBP (3- and 7-fold in Nonidet P-40 and octyl-beta-glucoside respectively). The receptor was isolated using recombinant RBP coupled to Reacti-Gel 6X affinity matrix. The purified material contained major and minor protein species of 63 and 55 kDa respectively on SDS/PAGE.
Sivaprasadarao
A
A
Department of Biochemistry and Molecular Biology, University of Leeds, U.K.
Boudjelal
M
M
Findlay
J B
JB
D001665:Binding Sites; D002846:Chromatography, Affinity; D003902:Detergents; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007457:Iodine Radioisotopes; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism; D011247:Pregnancy; D011257:Pregnancy Proteins / Q000302:isolation & purification* / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000302:isolation & purification* / Q000378:metabolism; D012177:Retinol-Binding Proteins / Q000378:metabolism*; D012995:Solubility
D003902:Detergents; D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D011257:Pregnancy Proteins; D011956:Receptors, Cell Surface; D012177:Retinol-Binding Proteins; C027928:retinol binding protein receptor