Tohoku J. Exp. Med., 2000, 192 (1)

Simple and Rapid Determination of GTPase Activity by Capillary Electrophoresis without Radioisotope

HIROKO KAWATA, KOICHI KURODA, YOKO ENDO,1 YOSHINORI INOUE and GINJI ENDO

Department of Preventive Medicine and Environmental Health, Osaka City University Medical School, Osaka 545-8585, and 1Department of Public Health, Kansai Medical University, Moriguchi 570-8506

  • In order to determine guanosine-5-triphosphatase (GTPase) activity, we developed a simple, rapid and reliable method that utilizes capillary electrophoresis without radioisotope. Tubulin-GTPase was used for simple measurement of GTPase activity utilizing capillary electrophoresis. Tubulin, a component of microtubules, was incubated with guanosine-5-triphosphate (GTP) in 100 mM 2-(N-morpholino) ethanesulfonic acid (MES) buffer (pH 6.5). Guanosine-5-diphosphate (GDP) was determined as the hydrolyzed product of GTP. Guanosine-5-monophosphate, GDP and GTP in the filtrate of the mixture were clearly separated using 10 mM MES buffer (pH 6.5) (migration time, 3.8, 5.5 and 7.2 minutes, respectively) with a fused-silica capillary column. The quantification of GDP was based on the peak area, which increased linearly with the concentration of GDP from 1 to 50 mM (r20.995). The peak area and migration time had good reproducibility; the intra-assay coefficient of variation (n6) was 1.3% for peak area and 0.6% for migration time. As an application of this method, we examined the effect of dimethylarsinic acid, an effective antimitotic agent, on tubulin-GTPase. Dimethylarsinic acid inhibited tubulin-GTPase activity in a dose-dependent manner. The inhibition was not complete and the maximum decrease of the activity was about 50% at 200 mM dimethylarsinic acid. Thus, since this method is clean, simple and rapid, its application to the study of various GTPase proteins is expected to be useful.
    Key words--- capillary electrophoresis; GTPase; tubulin; GDP; non-radioisotope
    © 2000 Tohoku University Medical Press

    Tohoku J. Exp. Med., 2000, 192, 67-79
    Address for reprints: Hiroko Kawata, Department of Preventive Medicine and Environment Health, Osaka City University Medical School, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
    e-mail: hirozou@med.osaka-cu.ac.jp

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