Continuous monitoring of P-i release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay

ATP and CTP are hydrolyzed during self-assembly of actin and tubulin, respectively. It is known that nucleotide is hydrolyzed on the polymer in two consecutive steps, chemical cleavage of the gamma-phosphate followed by the slower release of P-i. This last step has been shown to play a crucial role in the dynamics of actin filaments and microtubules. Thus far, evidence for a transient GDP-P-i state in microtubule assembly has been obtained using a glass fiber filter assay that had a poor time resolution [Melki, R., Carlier, M.-F., & Pantaloni, D. (1990) Biochemistry 29, 8921-8932]. We have used a new P-i assay [Webb, M. R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887], in which the purine phosphorylase catalyzes the phosphorolysis of 2-amino-6-mercapto-7-methylpurine ribonucleoside (MESG) into mercaptopurine and ribose phosphate, which is accompanied by an increase in absorbance. This enzyme-linked assay has been used to follow the release of P-i during polymerization of Mg-actin. A value of 350 s was found for the half-time for P-i release on F-actin, in good agreement with previous determinations. The release of P-i following GTP hydrolysis in microtubule assembly was followed using a slopped-flow apparatus. Rapid microtubule assembly was achieved using taxol. The use of a stopped-flow apparatus permitted the continuous recording, with a dead time of 0.8 ms, of both time courses of microtubule assembly and P-i release with greatly improved lime resolution. The release of P-i developed with a short lag (35 and 2 s for G-actin and tubulin, respectively) following assembly and appeared 50-fold faster on microtubules than on actin filaments.