Removal of β_III Isotype Enhances Taxol Induced Microtubule Assembly

抄録

The interaction of β_III-depleted tubulin with taxol was investigated. A monoclonal antibody against the β_III tubulin isotype was immobilized on a sepharose 4B column and used to remove the β_III tubulin isotype from unfractionated tubulin. The assembly of β_III-depleted tubulin in the presence of taxol was enhanced compared to unfractionated tubulin. The critical concentration of unfractionated tubulin in the presence of 10 μM taxol is 0.4 mg/ml, while the critical concentration of β_III-depleted tubulin is 0.16 mg/ml. At different concentrations of taxol, the assembly of β_III-depleted tubulin is increased relative to that of un fractionated tubulin and reaches the maximumat about a 1 : 1 ratio of tubulin and taxol. The assembly of un fractionated tubulin and β_III-depleted tubulin has also been studied by electron microscopy. After 2 minutes at 37°C, unfractionated tubulin assembly in the presence of 10 μM taxol results only in ribbon-like and ring structures; there are no visible microtubules. By 5 minutes, microtubules appear and increase in length. The assembly of β_III-depleted tubulin in the presence of 10 μM taxol occurs more quickly. In contrast to the case with un fractionated tubulin, β_III-depleted tubulin assembles within 2 minutes into microtubules which increase in length with time. At 30 minutes, microtubules assembled from β_III-depleted tubulin are shorter than the microtubules assembled from unfractionated tubulin. There is no visible difference between the microtubules assembled from unfractionated tubulin and β_III-depleted tubulin. Taxol-induced β_III-depleted tubulin assembly is more resistant to the inhibiting effect of podophyllotoxin and colchicine. It is also less sensitive to the inhibiting effect of cold temperature.