Thiol-dependent subunit interactions of a major fat cell plasma membrane glycoprotein.
A major glycoprotein fraction of the isolated adipocyte plasma membrane migrated in the 200,000-dalton region of dodecyl sulfate polyacrylamide gels following solubilization in 2% dodecyl sulfate/4M urea at room temperature in the absence of reductant. Limited heat treatment allowed resolution of the glycoprotein into two distinct bands in the same high-molecular-weight region plus a new 94,000-dalton glycoprotein band. Prolonged incubation of solubilized plasma membranes at 100 degrees C for 15-30 min or incubation with reductant resulted in complete conversion of the high-molecular-weight bands to the 94,000-dalton region, indicating dissociation of dimers to the monomeric form. When the dimer bands on column gels were electrophoresed in the second dimension on slab gels in the presence of reductant, no low-molecular-weight bands were observed other than that in the 94,000-dalton region. The effects of limited heat treatment to permit resolution of the two dimers and the extended treatment to convert the dimers to the monomeric form were markedly inhibited by alkylation of the solubilized membrane protein with N-ethylmaleimide or oxidation with H2O2 or diamide. However, these latter treatments did not prevent complete dissociation of dimers due to addition of reductants. These results suggest that two glycoprotein fractions may exist as dimers in the native fat cell plasma membrane. The data are consistent with a model in which the glycoprotein subunits are linked by hydrophobic bonds that are sensitive to reduction of intramolecular disulfides but are stabilized by alkylation or oxidation of the glycoprotein sulfhydryls.
D017667:Adipocytes / Q000648:ultrastructure*; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D046911:Macromolecular Substances; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism*
D046911:Macromolecular Substances; D008562:Membrane Glycoproteins; D013438:Sulfhydryl Compounds