Ultraviolet irradiation of the [3Hjcolchicine-tubulin complex leads to direct photolabeling of tubulin with low but practicable efficiency. The bulk (70% to >90%) of the labeling occurs on (8-tubulin and appears early after irradiation, whereas a-tubulin is labeled later. The labeling ratio of .8-tubulin to a-tubulin (13/a ratio) is reduced by prolonged incubation, prolonged irradiation, urea, high ionic strength, the use of aged tubulin, dilution of tubulin, or large concentrations of colchicine or podophyllotoxin. Glycerol increases the (3/a ratio. Limited data with (3Hlpodophyllotoxin show that it covalently bound with a similar fl/a distribution.Vinblastine, on the other hand, exhibits preferential attachment to a-tubulin. The possibilities that colchicine binds at the interface between a-tubulin and j3-tubulin, that the drug spans this interface, and that both subunits may contribute to the binding site are suggested.Despite the fact that colchicine has been used as an antimicrotubule agent for many years, there is no unanimity regarding the location of the high-affinity binding site for the drug in the tubulin dimer, which is formed by the noncovalent association ofthe similar but not identical q and (3 monomers.Several studies have assigned the site to the a-subunit, but uncertainties exist regarding the specificity of the' reactions used. Thus, N-bromoacetyldesacetylcolchicine showed nonspecific alkylation (1), photoaffinity labels used long spacer arms (2, 3), and studies with limited proteolysis could have been affected by rearrangements during proteolysis in the damaged protein (4). Colchicine binding to a site on ,f-tubulin has been proposed on the basis of indirect experiments dealing with the reactivity ofcysteine residues in ,3-tubulin (5) and by findings that most tubulin mutations that confer colchicine resistance occur in 83-tubulin genes (6-8).The excitation maximum of colchicine occurs at a higher wavelength than that of the tryptophan residues of tubulin; hence, direct photolabeling of tubulin with colchicine, without irradiating the protein, appeared to be feasible. However, stoichiometric covalent binding would not be expected for such a reaction because the efficiency of direct photolabeling tends to be <25% (9) because of the short colchicine fluorescence lifetime (of 1.14 ns) (10) with little intersystem crossing to the triplet state or long lifetimes (11), and because of the powerfully competing photoisomerization reaction to form lumicolchicines from excited-state colchicine, which causes dissociation of the ligand (12-14). Nevertheless, such a reaction might be less subject to the specificity problems noted above and thus increases the probability that colchicine will cross-link to the "correct" site. The following study explores the conditions for the direct photolabeling reaction, the localization of the covalently bound colchicine, and the factors influencing the distribution of the drug on tubulin. A portion of this material has been presented (15).
MATERIALS AND METHODST...
