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 microtubule assembly and alter microtubule protein composition (Pedrotti et al., 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 develop a simple purification procedure for MAP1A using twice-cycled microtubule protein prepared from whole bovine brain. A single chromatographic 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 manner to unpolymerized tubulin and assembled microtubules, (b) binds 13-15 mol of tubulin dimers in assembled microtubules, (c) promotes both 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 concentration for assembly, and MAP1A-promoted incorporation of dimers has an association rate constant (K+1) of 39.3 x 10(6) M-1s-1 and a dissociation rate constant (K-1) of 15 s-1; both constants are about 2-3-fold higher compared with MAP2.
Pedrotti
B
B
Department of Biology, University of Milan, Italy.
Islam
K
K
D000818:Animals; D001921:Brain / Q000648:ultrastructure; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D017239:Paclitaxel / Q000494:pharmacology; D011108:Polymers; D014404:Tubulin / Q000378:metabolism*
D046911:Macromolecular Substances; D008869:Microtubule-Associated Proteins; D011108:Polymers; D014404:Tubulin; D006160:Guanosine Triphosphate; D017239:Paclitaxel