IN-VITRO ANALYSIS OF MICROTUBULE ASSEMBLY OF ISOTYPICALLY PURE TUBULIN DIMERS - INTRINSIC DIFFERENCES IN THE ASSEMBLY PROPERTIES OF ALPHA-BETA-II, ALPHA-BETA-III, AND ALPHA-BETA-IV TUBULIN DIMERS IN THE ABSENCE OF MICROTUBULE-ASSOCIATED PROTEINS

Microtubule assembly of different beta tubulin isotypes in the presenc e of 4 m glycerol and 6 mm magnesium ion demonstrates significantly di fferent characteristics. alphabetaII and alphabetaIV assembled faster and to a greater extent than did unfractionated phosphocellulose-purif ied tubulin (PC-tubulin). Microtubule assembly from alphabetaIII showe d a distinctive delay in nucleation, proceeded at a slower rate than t hose of the other beta tubulin isotypes, and had the highest critical concentration. However, treatment of beta tubulin isotypes with subtil isin to remove the C-terminal domain of the tubulin dimer abolished th ese differences in microtubule assembly pattern and enhanced self-asse mbly. The kinetic analysis of microtubule elongation of different beta tubulin isotypes also showed significant differences. Elongation of a lphabetaIII from microtubule seeds had a lower apparent K(alpha) and a lower apparent K(d) than did alphabetaII and alphabetaIV. The dynamic behaviors of different beta tubulin isotypes were qualitatively simil ar to each other and fit the dynamic instability model. However, micro tubules formed from alphabetaIII appeared to be less dynamic than micr otubules formed from other beta tubulin isotypes. Our results suggest that the betaIII isotype might have a different conformation than do t he other beta tubulin isotypes. The distinctive nucleation and elongat ion behaviors of the alphabetaIII dimers demonstrated in vitro may hav e a significant influence on microtubule functions in vivo.