Affinity labeling of the sialoglycopeptide antimitogen receptor.
An 18-kDa 125I-sialoglycopeptide growth inhibitor was covalently cross-linked to its binding site on intact cultured Swiss 3T3 cells by three bifunctional cross-linkers with short (dimethyl adipimate), medium (disuccinimidyl suberate), and long (bis(2-succinimidooxycarbonyloxyethyl)sulfone) chain lengths. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography demonstrated a band of Mr approximately 168,000 regardless of which cross-linker was used. The labeling of this band was specific in that it was prevented by excess unlabeled inhibitor and the apparent molecular weight of the cross-linked receptor-ligand complex was unchanged by treatment with reducing agent. The efficiency of the cross-linking was increased by increasing pH, and the extent of covalent cross-linking was dependent on the concentration of the bifunctional reagent. Octyl glucoside and sodium dodecyl sulfate were effective in solubilizing the receptor while Triton X-100 did not extract the receptor from the plasma membrane. These observations suggest that the 168-kDa binding species represents the 125I-sialoglycopeptide cross-linked to a specific plasma membrane receptor and that the receptor does not appear to contain interchain disulfide bonds.
Sharifi
B G
BG
Division of Biology, Kansas State University, Manhattan 66506.
Johnson
T C
TC
D000345:Affinity Labels / Q000378:metabolism*; D000818:Animals; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004220:Disulfides / Q000032:analysis; D005960:Glucosides; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic / Q000378:metabolism*; D012795:Sialoglycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D012967:Sodium Dodecyl Sulfate; D012995:Solubility
D000345:Affinity Labels; D003432:Cross-Linking Reagents; D004220:Disulfides; D005960:Glucosides; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D011971:Receptors, Immunologic; D012795:Sialoglycoproteins; C032193:glycoprotein receptor GPIb-IX; C047908:sialoglycopeptide inhibitor protein, bovine; C018619:octyl-beta-D-glucoside; D012967:Sodium Dodecyl Sulfate