Localization of glycolytic enzymes in murine erythrocytes resealed in the presence of the cytoplasmic domain of murine band 3 (residues 1-398). Murine wild-type cdb3 was dialyzed against 5 mM of potassium phosphate containing 160 mM of sodium chloride, pH 7.4, and concentrated to 6.8 mg/mL using a centricon-30 (Millipore, Billerica, MA). Freshly drawn mouse erythrocytes were washed in the above buffer and resuspended at 50% hematocrit in the same buffer containing mouse cdb3. The suspension was introduced into mini dialysis units, and lysed and resealed as described by Campanella et al.1 After resealing, erythrocytes were fixed, permeabilized, and stained for GAPDH, aldolase, LDH, and PK and visualized with secondary antibody conjugated with Cy2 (492 510). Control erythrocytes were lysed and resealed in the absence of cdb3, but otherwise treated identically. Glycolytic enzymes are displaced from the membrane in cells containing resealed murine cdb3.
