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.
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 gamma-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.
Melki
R
R
Laboratoire d'Enzymologie et Biochimie Structurales, Centre National de la Recherche Scientifique, Gif-Sur-Yvette, France.
Fievez
S
S
Carlier
M F
MF
D000199:Actins / Q000737:chemistry / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001921:Brain; D006151:Guanosine / Q000031:analogs & derivatives*; D006160:Guanosine Triphosphate / Q000378:metabolism*; D007202:Indicators and Reagents; D007700:Kinetics; D046911:Macromolecular Substances; D018482:Muscle, Skeletal; D010710:Phosphates / Q000032:analysis / Q000378:metabolism*; D011683:Purine-Nucleoside Phosphorylase; D011817:Rabbits; D013552:Swine; D013872:Thionucleosides; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism*
D000199:Actins; D007202:Indicators and Reagents; D046911:Macromolecular Substances; D010710:Phosphates; D013872:Thionucleosides; D014404:Tubulin; D006151:Guanosine; C074696:6-mercapto-7-methylguanosine; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D011683:Purine-Nucleoside Phosphorylase