PURIFIED NATIVE MICROTUBULE-ASSOCIATED PROTEIN MAP1A - KINETICS OF MICROTUBULE ASSEMBLY AND MAP1A TUBULIN STOICHIOMETRY

In a recent study, we have shown that sulfonate buffers affect microtu bule assembly and alter microtubule protein composition (Pedrotti et a l., 1993). In particular, we noted that PIPES buffer leads to removal of MAP1 from the microtubule surface without affecting the association of MAP2 with microtubules. This observation has been exploited to dev elop a simple purification procedure for MAP1A using twice-cycled micr otubule protein prepared from whole bovine brain. A single chromatogra phic step on an ion-exchange column results in >90% pure MAP1A. Using purified MAP1A, we now show that MAP1A (a) binds in a dose-dependent m anner to unpolymerized tubulin and assembled microtubules, (b) binds 1 3-15 mol of tubulin dimers in assembled microtubules, (c) promotes bot h nucleation and elongation of tubulin, and (d) promotes incorporation of tubulin dimers at low GTP concentrations and of tubulin dimers and oligomers at high GTP concentrations. MAP1A lowers the critical conce ntration for assembly, and MAP1A-promoted incorporation of dimers has an association rate constant (K-+1) of 39.3 x 10(6) M(-1)s(-1) and a d issociation rate constant (K--1) of 15 s(-1); both constants are about 2-3-fold higher compared with MAP2.