guanosine-triphosphate and 6-mercapto-7-methylguanosine

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.

Topics: Actins; Adenosine Triphosphate; Animals; Brain; Guanosine; Guanosine Triphosphate; Indicators and Reagents; Kinetics; Macromolecular Substances; Muscle, Skeletal; Phosphates; Purine-Nucleoside Phosphorylase; Rabbits; Swine; Thionucleosides; Tubulin

The mechanism of GTPase-activating protein (GAP) activation of p21ras GTP hydrolysis has been investigated by measuring the kinetics of release of Pi during the hydrolysis. The measurement uses a continuous spectroscopic assay for Pi, based on a guanosine analogue, 2-amino-6-mercapto-7-methylpurine ribonucleoside, as substrate for purine nucleoside phosphorylase [Webb, M.R. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 4884-4887]. This phosphorolysis gives an absorbance increase at 360 nm, so that when the reaction is coupled to GTP hydrolysis, the change in absorbance gives the total amount of Pi released from the p21ras. The rate of the absorbance increase gives the GTPase activity. This provides a non-radioactive method of determining p21ras concentration and GAP activity. It was used to determine the interaction of GAP with wild-type p21ras and two mutants (Leu-61/Ser-186 and Asp-12), all in the GTP (or guanosine 5'-[ beta gamma-imido]triphosphate) form. The Leu-61/Ser-186 mutant binds 10-fold tighter than does the wild-type protein. The Asp-12 mutant binds to GAP with the same affinity as the wild-type protein. A novel GTPase activity was characterized whereby the EDTA-induced nucleotide release and GAP-activated cleavage of bound GTP leads to steady-state turnover of GTP hydrolysis. An assay for GAP is described based on this activity.

Topics: Ammonium Sulfate; Edetic Acid; GTPase-Activating Proteins; Guanosine; Guanosine Triphosphate; Hydrolysis; Kinetics; Phosphates; Proteins; Proto-Oncogene Proteins p21(ras); Purine-Nucleoside Phosphorylase; ras GTPase-Activating Proteins; Spectrophotometry, Ultraviolet; Thionucleosides