Properties of purified colchicine-binding protein from a cultured carrot cell extract.

Abstract

Colchicine-binding protein (CBP) was purified from a cultured carrot cell extract by DEAE-Sephacel, phosphocellulose and Sephadex G200 column chromatographies. The purified CBP separated into three bands on SDS-polyacrylamide gel electrophoresis. One of them reacted with a monoclonal antibody against chick brain alpha-tubulin and the other two with that against beta-tubulin. Colchicine-binding activity of the purified protein was enhanced by tartrate and inhibited little by an excess of podophyllotoxin. It decayed following first order kinetics, but was more stable than the CBP in the crude extract. The binding constant of the purified CBP for colchicine was 0.57 microM-1 and the number of binding sites of colchicine per mg protein was about 2 nmol. This binding constant is about ten times lower than that of porcine brain tubulin under identical conditions.