Crosslinking of the Glycoproteins in Human Erythrocyte Membranes

Abstract

The glycoproteins of human erythrocyte membranes were crosslinked with dimethyl adipimidate dihydrochloride. On sodium dodecyl sulfate-polyacrylamide gel electrophoregrams of the crosslinked solubilized membranes, at least three new glycoprotein complexes appeared in addition to the normal glycoprotein species. One of the new glycoprotein complexes was shown to contain two of the three species of membrane glycopro. teins. Glycoproteins have been shown to be vital in determining the structure and organization of plasma membranes (1-4), and there is evidence suggesting their important role in cell-to-cell contact, adhesion, hormone interaction, and viral transformation (5-12). Little is known concerning the structures of membrane glycoproteins in animal cells, with the exception of glycoproteins from human erythrocyte membranes.Three major species of glycoproteins have been found in human erythrocyte membranes (13); one of these has been implicated in reversible aggregation and dispersion in the membrane plane (2-4). Interactions with other membrane components may be important in determining their lateral movement and, in fact, there are such indications (4). Chemical crosslinking of the glycoproteins to adjacent molecules is one approach to studying their organization and interactions in the membrane. A variety of bifunctional crosslinking reagents were tested for that purpose. None of these reagents was successful in crosslinking glycoproteins, although most or all of the proteins in the membrane have been crosslinked by this technique (14-16). It will be shown here that the glycoproteins and proteins can be crosslinked with dimethyl adipimidate dihydrochloride, and that new glycoprotein complexes can be produced. MATERIALS AND METHODSOut-dated, normal human blood was purchased from the Wyoming Blood Service, Cheyenne, Wyo. Erythrocyte ghosts were prepared as described (17).Dimethyl adipimidate dihydrochloride (DIMA) was prepared according to the method of McElvain and Schroeder (18) and dissolved in a buffer containing 0.9 g/100 ml of NaCl and 5 mM Na2HPO4 (pH 7.5). The erythrocyte ghosts and DMA were mixed in the buffer to make the concentration of ghost proteins 0.15 mg/ml and reagent 0.5 mg/ml. The protein concentration was determined by the modified Lowry method (19). The reaction was stopped by centrifugation and resuspension of the ghosts twice in the buffer.The membrane proteins were solubilized in 1% sodium dodecyl sulfate (SDS), subjected to electrophoresis in SDSpolyacrylamide gels, and stained for either glycoproteins or proteins, as described (20). All of the chemicals necessary to prepare the gels were purchased from the Eastman Kodak Co. The gels were scanned on an ISCO gel scanner at 550 nm.The crosslinking was cleaved by ammonolysis as follows: A zone corresponding to a specific band of crosslinked product in an unstained duplicate gel was cut into about 2-mm pieces and incubated in 100 ,ul of a solution containing 35% ammonium hydroxide, 10% SDS, acetic acid, and 2-mercaptoethanol...

Changes in lipid ordering and state of aggregation in lymphocyte plasma membranes after exposure to mitogens

“…The glycosphingolipids could also be associated with membrane proteins and glycoproteins, bearing receptors either by lateral hydrogen bonding or interaction between sugar residues. Evidence for such an association in the human red cell membrane has been presented by Ji (1974). Under these circumstances, ligandinduced cross-linking of receptor-bearing proteins would aggregate the glycosphingolipids.…”

“…Following cell activation, a number of early changes in the membrane and membrane mediated events have been reported. These include an influx of K § (Quastel & Kaplan, 1970), Ca § § (Allwood et al, 1970, Whitney & Sutherland, 1973, amino acids (van den Berg & Betel, 1973, 1974, glucose and uridine (Peters & Hausen, 1971) as well as increases in membrane phospholipid metabolism (Fisher & Mueller, 1971, Masuzawa et al, 1973. None of these studies, however, throw any light on how combination of a mitogen with the cell surface initiates these changes.…”

Studies of Glutamate Dehydrogenase

“…Hucho et al [ 101 investigated the symmetry of some tetrameric enzymes consisting of identical protomers with dimethylimidates. With the aim of identifying nearest neighbors the cross-linking technique has since been extended to more complex systems such as ribosomes [ l l -131, membranes [14,15] and nuclei [16,17].…”