Kato T, Kakiuchi M, Okamura S
J Biochem. 1985 Aug;98(2):371-7. doi: 10.1093/oxfordjournals.jbchem.a135291.
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.
秋水仙碱结合蛋白（CBP）通过DEAE-葡聚糖凝胶、磷酸纤维素和葡聚糖G200柱色谱从培养的胡萝卜细胞提取物中纯化得到。纯化后的CBP在SDS-聚丙烯酰胺凝胶电泳上分离成三条带。其中一条与抗鸡脑α-微管蛋白的单克隆抗体反应，另外两条与抗β-微管蛋白的单克隆抗体反应。纯化蛋白的秋水仙碱结合活性被酒石酸盐增强，且几乎不受过量鬼臼毒素的抑制。它遵循一级动力学衰减，但比粗提取物中的CBP更稳定。纯化后的CBP与秋水仙碱的结合常数为0.57μM-1，每毫克蛋白质的秋水仙碱结合位点数约为2 nmol。在相同条件下，该结合常数约比猪脑微管蛋白的结合常数低十倍。
