REMOVAL OF BETA(III) ISOTYPE ENHANCES TAXOL-INDUCED MICROTUBULE ASSEMBLY

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 muM taxol is 0.4 mg/ml, while the critical concentration of beta(III)-depleted tubulin is 0.16 mg/ml. At different concentrations 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 muM 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 muM 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.