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A Purification and In Vitro Activity Assay for a pppGpp Synthetase from Clostridium difficile
Published on: November 3, 2018
A Purification and In Vitro Activity Assay for a (p)ppGpp Synthetase from Clostridium difficile
Astha Pokhrel1, Asia Poudel1, Erin B Purcell2
1Department of Chemistry and Biochemistry, Old Dominion University.
Abstract:
Kinase and pyrophosphokinase enzymes transfer the gamma phosphate or the beta-gamma pyrophosphate moiety from nucleotide triphosphate precursors to substrates to create phosphorylated products. The use of γ-32-P labeled NTP precursors allows simultaneous monitoring of substrate utilization and product formation by radiography. Thin layer chromatography (TLC) on cellulose plates allows rapid separation and sensitive quantification of substrate and product. We present a method for utilizing the thin-layer chromatography to assay the pyrophosphokinase activity of a purified (p)ppGpp synthetase. This method has previously been used to characterize the activity of cyclic nucleotide and dinucleotide synthetases and is broadly suitable for characterizing the activity of any enzyme that hydrolyzes a nucleotide triphosphate bond or transfers a terminal phosphate from a phosphate donor to another molecule.
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