Simple and rapid determination of Gtpase activity by capillary electrophoresis without radioisotope.
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 microM (r2=0.995). The peak area and migration time had good reproducibility; the intra-assay coefficient of variation (n=6) 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 microM 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.
Kawata
H
H
Department of Preventive Medicine and Environmental Health, Osaka City University Medical School, Japan. hirozou@med.osaka-cu.ac.jp
Kuroda
K
K
Endo
Y
Y
Inoue
Y
Y
Endo
G
G
D002021:Buffers; D002101:Cacodylic Acid / Q000494:pharmacology; D019075:Electrophoresis, Capillary; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000378:metabolism; D014404:Tubulin / Q000378:metabolism
D002021:Buffers; D014404:Tubulin; D006153:Guanosine Diphosphate; D002101:Cacodylic Acid; D020558:GTP Phosphohydrolases