Microtubular Proteins in Pigeon Erythrocyte Membranes

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Abstract
[3H]Colchicine binds in a concentration and temperature dependent, saturatable and noncooperative manner to cytoplasmic proteins from pigeon erythrocytes: Kd = 3.5 .times. 10-7 M at 37.degree. C. Binding of [3H]colchicine at 0.degree. C and of [3H]lumicolchicine at 37.degree. C was significantly reduced. This shows that microtubular proteins are present in the cytoplasm of pigeon erythrocytes. Antibody against bovine brain tubulin was raised in rabbits and confirmed by immunodiffusion, passive immunohemolysis and in radioimmunoassay. Pigeon erythrocyte membrane proteins solubilized with 2% sodium cholate competed with 125I-labeled tubulin in the radioimmunoassay, although much higher concentrations of membrane proteins than of purified bovine brain tubulin were required for effective competition. No binding to antibody occurred with boiled solubilized membrane preparations. Similar results were obtained with antitubulin-dependent passive immunohemolysis of tubulin-coated sheep erythrocytes in the presence of complement. The presence of tubulin in membranes was verified by binding intact pigeon erythrocytes to colchicine-Sepharose beads at 37.degree. C. Free colchicine (5 mM) or incubation at 0.degree. C prevented binding. Lumicolchicine-Sepharose beads did not attach to erythrocytes at 37.degree. C.