In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis.
A capillary electrophoresis (CE)-based method for the in vitro detection and monitoring of nucleotide-triphosphatase activity is described. This robust and reproducible method was used to investigate GTPase activity of a recombinant protein construct containing the catalytic domain of Human SEPT4/Bradeion beta (GST-rDGTPase). This example application demonstrates that the CE technique can replace classical radioactive methods for GTPase activity assays and may be used as a routine analytical tool. Enzyme kinetics of GST-rDGTPase was studied and yielded the following kinetic parameters: v(max) = 1.7 microM min(-1) +/- 0.1, Km = 1.0 mM +/- 0.3, and apKcat = 9 x 10(-3) s(-1). In addition the effect of co-factors such as Mg2+ and Mn2+ on the catalytic activity was investigated. The described analytical method was also shown to be useful to analyze diphosphated and triphosphated forms of other nucleotides.
Hillebrand
Sandro
S
Centro de Biotecnologia Molecular e Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, São Carlos, SP, Brazil.
Garcia
Wanius
W
Delmar Cantú
Marcelo
M
de Araújo
Ana Paula Ulian
AP
Tanaka
Manami
M
Tanaka
Tomoo
T
Garratt
Richard Charles
RC
Carrilho
Emanuel
E
D015374:Biosensing Techniques; D002384:Catalysis; D002413:Cations, Divalent; D019075:Electrophoresis, Capillary / Q000379:methods*; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008904:Miniaturization; D009841:Oligonucleotides / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism; D013379:Substrate Specificity; D013997:Time Factors
D002413:Cations, Divalent; D009841:Oligonucleotides; D010710:Phosphates; D008345:Manganese; D020558:GTP Phosphohydrolases; D008274:Magnesium