Cross-linking of intact erythrocyte membrane with a newly synthesized cleavable bifunctional reagent.

In order to clarify the interaction of membrane proteins in intact cells (not ghosts), a new bifunctional cleavable reagent, maleimidomethyl-3-maleimido propionate (MMP), was synthesized. Human intact erythrocytes and ghosts were incubated with this reagent. Cross-linked products were analyzed by two-dimensional polyacrylamide gel electrophoresis with incubation of the first dimensional gel in 0.1 N NaOH for 15 min at room temperature before the second run. In the case of ghosts, newly appeared bands were tentatively identified as a spectrin dimer, a complex of band 2 with band 5 degrees, which runs very slightly more slowly than band 5 and is phosphorylated by 32P in intact cells, a complex of band 3 with band 5, a dimer of band 3 and a dimer of band 5 degrees. In the case of intact cells, the newly appeared bands were complexes of band 3 with bands 4.3, 4.5, 4.6, and 4.7 and band 5 dimer, in addition to those seen in the case of ghosts. The technique of membrane phosphorylation permitted the clearer identification of each band. This reagent is useful for studying the relationship among cell membrane proteins in intact erythrocytes.