The binding of Zn2+ to tubulin and the ability of this cation to promote the polymorphic assembly of the protein were examined. Equilibrium binding showed the existence of more than 60 potential Zn2+ binding sites on the dimer, including a number of high-affinity sites. The number of high-affinity sites, estimated by using a standard amount of phosphocellulose to remove more weakly bound Zn2+, reached a maximum of 6-7.5 with increasing levels of Zn2+ in the incubation solution. The number also increased with time of incubation at a single Zn2+ concentration. It is suggested that tubulin is slowly denatured in the presence of Zn2+, exposing more binding sites. Cu+ and Cd2+ were effective inhibitors of Zn2+ binding; Mg2+, Mn2+, and Co2+ were much less effective, and Ca2+ was without effect. Zn2+ does not replace the tightly bound Mg2+. GTP reduces the amount of Zn2+ binding under equilibrium conditions and the amount bound to high-affinity sites. Zinc-induced protofilament sheets are produced at a Zn2+/tubulin ratio of 5 in the presence of 0.5 mM GTP, conditions where about two to three Zn2+ ions would be bound to the dimer. At higher GTP concentrations, less assembly occurred, and the products were narrower sheets and microtubules. Zn2+-tubulin, isolated from phosphocellulose, will not assemble unless Mg2+ and dimethyl sulfoxide (Me2SO) or more Zn2+ is added. Broad protofilament sheets, formed from Zn2+-tubulin in the presence of Mg2+ and Me2SO, contain slightly more than one Zn2+ per dimer. It is concluded that Zn2+ stimulates tubulin assembly by binding directly to the protein, not via a ZnGTP complex.

Irradiation of tubulin in the presence of [3H]GTP or [3H]GDP at 254 nm led to the covalent incorporation of nucleotide into the protein. The specific nature of the labeling was shown in the following manner: with tubulin depleted of exchangeable nucleotide, the amount of labeling increased to a plateau value as the [3H]GTP concentration was increased, with saturation being reached at a ratio of approximately 1.5; the same amount of labeling was obtained with GTP/tubulin ratios of 1 and 100; [3H]GMP was not incorporated into the dimer, nor did GMP inhibit the incorporation of [3H]GTP; [3H]ATP was not incorporated; [3H]GTP incorporation did not occur into denatured tubulin or into serum albumin. When [alpha-32P]GTP was used in the irradiation experiments, sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the carboxymethylated protein demonstrated that the incorporated label was associated with the beta subunit. The radiation treatment did cause changes in the tubulin molecule resulting in a decrease in assembly competence and in sulfhydryl groups, but these effects were minimized when a large excess of GTP was present during irradiation. Labeling of tubulin in the assembled state was much less than that observed in the free state.

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