Use of a GTP photoaffinity probe to resolve aspects of the mechanism of tubulin polymerization

Abstract

8-Azidoguanosine 5'-triphosphate (8-N3GTP) was used in a photoactivatable probe to examine the role of GTP in microtubule assembly. 8-N3GTP was able to substitute for GTP in the promotion of tubulin polymerization and was hydrolyzed at 37 degrees C in the presence or absence of colchicine or calcium. Photolysis of the analog in the presence of microtubular protein resulted in its covalent incorporation onto a GTP-specific site of the beta monomer. The efficiency of this incorporation was different when 8-N3GDP (which does not affect polymerization) was used in place of 8-N3GTP, implying a different orientation of the nucleoside diphosphate within the receptor site. During microtubule assembly, 8-N3GTP was hydrolyzed in situ at the tubulin-GTP exchangeable site in a process that was dependent upon polymerization. The use of [beta, gamma-32P]8-N3GTP and [gamma-32P]8-N3GTP indicated that this hydrolysis occurred concurrently with polymerization and that only nucleoside diphosphate remained bound to the polymerized tubulin.