Properties of Colchicine Binding Protein from Chick Embryo Brain. Interactions with Vinca Alkaloids and Podophyllotoxin

Abstract

The 120, 000 molecular weight colchicine binding protein found in the 100,000g supernatant fraction of chick embryo brain homogenates is considered to be a dimer subunit of brain microtubules. The colchicine binding activity of the protein was markedly influenced by the experimental conditions, and by other chemical agents which disrupt microtubules. Maximum colchicine binding activity was obtained at pH 6.7–6.8. A shift in the pH from this region exerted no effect on the protein–colchicine association reaction, but greatly influenced the binding activity of the protein indirectly, by increasing the inactivation constant of the protein. Ionic strength also affected the inactivation process. Binding activity was least stable at low ionic strength. Stability was increased by the addition of sodium salts; optimal stability was obtained with approximately 200 mM sodium glutamate. As in the case of pH, changes in ionic strength did not influence the protein–colchicine association reaction. The vinca alkaloids, vinblastine and vincristine, stabilized colchicine binding activity, but did not appear to influence the colchicine–protein association reaction. Stabilization was proportional to the logarithm of the vinca alkaloid concentration and occurred in a concentration range in which these agents produce their disruptive effects on microtubules. Vinblastine stabilized the binding activity of the protein to the same degree, whether or not colchicine was bound to the protein. With high concentrations of vincristine, nearly complete stabilization of colchicine binding activity was observed. In contrast to the vinca alkaloids, podophyllotoxin inhibited the binding of colchicine to the protein. Podophyllotoxin exerted no inhibitory effect when tested on preformed protein–colchicine complex. Podophyllotoxin did not inhibit the binding of colchicine by increasing the inactivation constant of the binding activity. Rather, an increase in stability of the binding activity was observed, even when podophyllotoxin was added to protein which already had the colchicine binding site(s) occupied. Therefore, the inhibitory effects of podophyllotoxin on the binding of colchicine may not be brought about by a simple competitive mechanism. © 1970, American Chemical Society. All rights reserved.