Photoaffinity labeling of tubulin with (2-nitro-4-azidophenyl)deacetylcolchicine: direct evidence for two colchicine binding sites

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Abstract
A new photoaffinity analogue of colchicine, (2-nitro-4-azidophenyl)deacetylcolchicine (NAP-DAC), bound to two classes of sites on bovine renal tubulin and photolabeled both the .alpha.- and .beta.-subunits. The apparent Ki for the photoaffinity analogue was 1.40 .+-. 0.17 .mu.M (mean .+-. SD, n = 3) as measured by competition with [3H]colchicine. Values of the apparent Kds for the two sites, as measured by the direct binding of the [3H]NAPDAC to tubulin, were 0.48 .+-. 0.11 .mu.M and 11.6 .+-. 3.5 .mu.M (mean .+-. SD, n = 6), and the corresponding stoichiometries of binding of the two sites were 0.25 .+-. 0.06 and 1.3 .+-. 0.4 mol/mol of tubulin (mean .+-. SD, n = 6). NAPDAC was a potent inhibitor of microtubule formation as detected by electron microscopy. When tubulin was photolabeled with NAPDAC at 25.degree. C, 15 .+-. 3 mol % (mean .+-. SD, n = 6) of the [3H]NAPDAC was covalently bound to the .alpha.-subunit, and 67 .+-. 9 mol % (mean .+-. SD, n = 6) was covalently bound to the .beta.-subunit. Since NAPDAC is a mixture of two interconvertible diastereomers, the photoincorporation of each was also examined. One diastereomer photolabeled both .alpha.- and .beta.-tubulin; however, the other did not significantly photolabel either subunit. Tubulin photolabeled with NAPDAC (1:1 mole ratio) exhibited a 23% decrease in colchicine binding. Preblocking and prephotolysis experiments with colchicine, NAPDAC, or ANPAH-CLC [Williams et al. (1985) J. Biol. Chem. 260, 13794-13802] provided evidence for conformational changes in tubulin upon colchicine binding. Peptide maps of [3H]NAPDAC-labeled .alpha.- and .beta.-tubulin, using Staphylococcus aureus V8 protease, demonstrated the presence of NAPDAC in one peptide of the .alpha.-subunit and in five peptides of the .beta.-subunit as detected by autoradiography. NAPDAC provides the first direct evidence for two colchicine binding sites on tubulin.