Residual nucleotide and tubulin's ability to polymerize with nucleotide analogs.
The nucleotide requirements for microtubule assembly following two different methods of nucleotide removal were defined. Microtubular protein extracted with charcoal in a buffer containing 1 mM EDTA showed an absolute dependence on GTP for assembly, whereas, microtubular protein extracted with charcoal in a buffer containing 1 mM MgCl2 could assemble with the nucleotide analogs, guanosine-5'-(beta,gamma-imido)triphosphate (GMP-PNP) and guanosine-5'-(beta-gamma-methylene)triphosphate. Assembly promoted by the nucleotide analogs proceeded at slower rate and to a lesser extent than the assembly induced by GTP. Furthermore, after the analog-induced assembly had reached a steady state level, the addition of GTP immediately induced further tubulin assembly to the level produced by GTP alone. Microtubules assembled with GMP-PNP or GTP contained approximately 2.8 mol of bound nucleotide per mol of dimer. The two different methods of charcoal extraction produced microtubular protein preparations that differed in their residual nucleotide and Mg2+ content. The EDTA-charcoal-extracted microtubular protein retained 1 mol of tightly bound nucleotide (GDP) and 1 mol of Mg2+ per mol of dimer, whereas, the Mg2+ charcoal-extracted microtubular protein retained 1.4 mol of bound nucleotide (1.0 mol of GDP + 0.4 mol of GTP) and 1.5 mol of Mg2+ per mol of dimer. The results suggest that only that fraction of tubulin subunits containing bound GTP can assemble with the nucleotide analogs and that microtubules may possess a third or polymer binding site for nucleotide.
D000818:Animals; D001921:Brain / Q000378:metabolism; D002942:Circular Dichroism; D006150:Guanine Nucleotides / Q000494:pharmacology*; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008854:Microscopy, Electron; D008870:Microtubules / Q000648:ultrastructure*; D011487:Protein Conformation; D013552:Swine; D014404:Tubulin / Q000378:metabolism*
D006150:Guanine Nucleotides; D014404:Tubulin; D008274:Magnesium