Removal of beta III isotype enhances taxol induced microtubule assembly.

Abstract

The interaction of beta III-depleted tubulin with taxol was investigated. A monoclonal antibody against the beta III tubulin isotype was immobilized on a sepharose 4B column and used to remove the beta III tubulin isotype from unfractionated tubulin. The assembly of beta 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 microM taxol is 0.4 mg/ml, while the critical concentration of beta III-depleted tubulin is 0.16 mg/ml. At different concentration of taxol, the assembly of beta III-depleted tubulin is increased relative to that of unfractionated tubulin and reaches the maximum at about a 1:1 ratio of tubulin and taxol. The assembly of unfractionated tubulin and beta III-depleted tubulin has also been studied by electron microscopy. After 2 minutes at 37 degrees C, unfractionated tubulin assembly in the presence of 10 microM 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 beta III-depleted tubulin in the presence of 10 microM taxol occurs more quickly. In contrast to the case with unfractionated tubulin, beta III-depleted tubulin assembles within 2 minutes into microtubules which increase in length with time. At 30 minutes, microtubules assembled from beta 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 beta III-depleted tubulin. Taxol-induced beta 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.