In vitro monitoring of GTPase activity and enzyme kinetics studies using capillary electrophoresis

Hillebrand, Sandro; Garcia, Wanius; Delmar Cantú, Marcelo; De Araújo, Ana Paula Ulian; Tanaka, Manami; Tanaka, Tomoo; Garratt, Richard Charles; Carrilho, Emanuel
September 2005
Analytical & Bioanalytical Chemistry;Sep2005, Vol. 383 Issue 1, p92
Academic Journal
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 β (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 μM min−1 ± 0.1, Km = 1.0 mM ± 0.3, and apKcat = 9 × 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.


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