Limited proteolysis of tubulin and the localization of the binding site for colchicine.

Limited proteolysis of porcine brain tubulin with trypsin resulted in a gradual loss of its colchicine binding activity as well as its ability of assembly into microtubules. The analysis of the tryptic degradation products showed a preferential proteolysis of alpha-tubulin subunit. This enzymatic proteolysis cleaved tubulin in one major site producing fragments of 36,000 and 16,000 daltons, the smaller polypeptides containing the carboxyl-terminal residue as shown by 14C tyrosination . However, proteolysis after incubation with 1 X 10(-3) M colchicine resulted in formation of the indicated fragments plus a 41,000-dalton fragment and smaller size peptides indicating the drug induces a second cleavage site closer to the carboxyl-terminal alpha-tubulin. Preincubation of tubulin with [3H]colchicine followed by proteolysis and separation of the fragments by Sephadex G-75 chromatography showed radioactive colchicine associated with the 16,000-dalton fragment and to the smaller size peptides resulting from digestion in the presence of the drug. The data indicate a localization of the colchicine-binding site in the 16,000-dalton segment containing the COOH-terminal region of alpha-tubulin subunit.