A capillary electrophoresis method for the characterization of ecto-nucleoside triphosphate diphosphohydrolases (NTPDases) and the analysis of inhibitors by in-capillary enzymatic microreaction.

Purinergic Signalling
Jamshed IqbalChrista E Müller

Abstract

A capillary electrophoresis (CE) method for the characterization of recombinant NTPDases 1, 2, and 3, and for assaying NTPDase inhibitors has been developed performing the enzymatic reaction within the capillary. After hydrodynamic injection of plugs of substrate solution with or without inhibitor in reaction buffer, followed by a suspension of an enzyme-containing membrane preparation, and subsequent injection of another plug of substrate solution with or without inhibitor, the reaction took place close to the capillary inlet. After 5 min, the electrophoretic separation of the reaction products was initiated by applying a constant current of -60 muA. The method employing a polyacrylamide-coated capillary and reverse polarity mode provided baseline resolution of substrates and products within a short separation time of less than 7 min. A 50 mM phosphate buffer (pH 6.5) was used for the separations and the products were detected by their UV absorbance at 210 nm. The Michaelis-Menten constants (K (m)) for the recombinant rat NTPDases 1, 2, and 3 obtained with this method were consistent with previously reported data. The inhibition studies revealed pronounced differences in the potency of reactive blue 2, pyridoxalphosphate-6-azo...Continue Reading

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Citations

Apr 1, 2009·Purinergic Signalling·David G ShirleyJean Sévigny
Sep 21, 2011·The Journal of Physiological Sciences : JPS·Takuto FujiiHideki Sakai
Aug 31, 2007·Biochemical Pharmacology·Mercedes N MunkondaJean Sévigny
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Feb 9, 2017·MedChemComm·Sang-Yong Lee, Christa E Müller
Aug 14, 2020·Journal of Medicinal Chemistry·Jenna L JeffreyJay P Powers
Dec 15, 2021·Journal of Neurochemistry·Marie GleizesLionel G Nowak

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Methods Mentioned

BETA
electrophoresis
transfection

Software Mentioned

GraphPad
ACE MDQ
PRISM

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