Continuous Monitoring of Pi 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

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Abstract
ATP and GTP 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 γ-phosphate followed by the slower release of Pi. 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−Pi 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 Pi 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 Pi during polymerization of Mg−actin. A value of 350 s was found for the half-time for Pi release on F-actin, in good agreement with previous determinations. The release of Pi following GTP hydrolysis in microtubule assembly was followed using a stopped-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 Pi release with greatly improved time resolution. The release of Pi 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.