Barry Zeeberg

Stoichiometry for guanine nucleotide binding to tubulin under polymerizing and nonpolymerizing conditions

Abstract The maximal stoichiometry for [ 3 H]GTP binding to depolymerized tubulin with saturating amounts of added [ 3 H]GTP is 0.4 mol/110,000 g protein. In contrast, 1 mol of radioactive nucleotide is incorporated into microtubules as a result of polymerization with [ 3 H]GTP. The different stoichiometries result from a difference in the nucleotide binding properties of ring protein under polymerizing and nonpolymerizing conditions: ring protein at 0 °C is devoid of binding activity but binds added radioactive guanine nucleotide during microtubule assembly. The radioactive nucleotide which is incorporated into rings during microtubule assembly is not displaced by excess GDP, although it is at a site which is distinct from the N site.

Preparation and characterization of [3H]ethyltubulin

Abstract Pure [ 3 H]ethyltubulin dimer, containing 1.07 mol of [ 3 H]ethyl groups/110.000 g protein was prepared by reaction of tubulin with acetaldehyde and [ 3 H]sodium borohydride. The derivatized tubulin dimer was shown to be native by the following criteria: (1) the stoichiometry for [ 3 H]GDP binding was similar to that for native tubulin: (2) it repeatedly copolymerized and codepolymerized with native tubulin with constant specific activity. The potential utility for [ 3 H]ethyltubulin in quantitating tubulin in biological samples by isotope dilution, and in studying the relationships between microtubules, rings, and dimers is discussed.