Characterization of the human placental membrane receptor for transcobalamin II-cobalamin.
A specific receptor on the plasma membrane of mammalian cells facilitates the uptake of vitamin B12 (cobalamin, Cbl) by receptor-mediated endocytosis of transcobalamin II-bound Cbl (TCII-Cbl). Purification of this receptor has proven to be difficult because of the lability of the protein during solubilization. Using human placental membranes as the source of the receptor, we have investigated alternative methods for solubilization of this protein and characterized a number of functional and structural properties. Homogenized and washed placental membranes show specific, saturable binding of TCII-Cbl with a Ka of 0.26 nM-1. Following solubilization of the membranes in 3-[(3-cholamidopropyl)-dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), the most efficient nonionic detergent tested, 21% of the receptor activity remained with the residual insoluble membrane fraction, a property of membrane proteins that are bound tightly to the cell cytoskeleton. Whereas 10 mM Chapso removed 79% of the receptor activity from the membrane preparation, only 3.7% of the TCII-Cbl binding activity was recovered in the solubilized fraction. The unstable TCII-Cbl binding in the soluble fraction was protected by the addition of 15% glycerol to the preparation and storage at -20 degrees C. The apparent M(r) of the receptor estimated by SDS-PAGE of the crosslinked receptor 125I-TCII-Cbl is approximately 58,000. The decrease in M(r) following digestion with several glycosidases and neuraminidase indicates that approximately 29% of the protein is carbohydrate which accounts for a core polypeptide of 41 kDa. Selective binding to a battery of lectins has established that the carbohydrate moiety of the receptor contains a large proportion of N-acetylglucosamine and terminal X-linked mannose.
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Division of Hematology/Oncology, Veterans Affairs Medical Center, Brooklyn, New York 11209.
Sai
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Rothenberg
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D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D005260:Female; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008970:Molecular Weight; D010920:Placenta / Q000378:metabolism*; D011247:Pregnancy; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D014155:Transcobalamins / Q000378:metabolism*; D014805:Vitamin B 12 / Q000378:metabolism*
D011956:Receptors, Cell Surface; D014155:Transcobalamins; C028176:transcobalamin receptor; D014805:Vitamin B 12