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 fo llowed 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 microtubule s. Thus far, evidence for a transient GDP-P-i state in microtubule ass embly has been obtained using a glass fiber filter assay that had a po or 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 pur ine phosphorylase catalyzes the phosphorolysis of 2-amino-6-mercapto-7 -methylpurine ribonucleoside (MESG) into mercaptopurine and ribose pho sphate, which is accompanied by an increase in absorbance. This enzyme -linked assay has been used to follow the release of P-i during polyme rization 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 determination s. The release of P-i following GTP hydrolysis in microtubule assembly was followed using a slopped-flow apparatus. Rapid microtubule assemb ly was achieved using taxol. The use of a stopped-flow apparatus permi tted the continuous recording, with a dead time of 0.8 ms, of both tim e courses of microtubule assembly and P-i release with greatly improve d lime resolution. The release of P-i developed with a short lag (35 a nd 2 s for G-actin and tubulin, respectively) following assembly and a ppeared 50-fold faster on microtubules than on actin filaments.