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Microtiter assay for glutamine synthetase biosynthetic activity using inorganic phosphate detection
Jeffrey D Gawronski1, David R Benson
1Department of Molecular and Cell Biology, University of Connecticut, Storrs, CT 06269-3125, USA.
Abstract:
A microtiter assay was developed for the improved detection of inorganic phosphate released from adenosine 5'-triphosphate (ATP) in the glutamine synthetase biosynthetic assay. In this assay, ascorbic acid replaces the traditionally used ferrous sulfate to reduce the phosphomolybdate complex. As a result, increased color development, linearity, and sensitivity are achieved. Additionally, in the microtiter format, multiple sets of kinetic experiments can be rapidly performed in parallel. The color that forms is rendered highly stable by the addition of sodium citrate. However, the commonly used sodium arsenite in this solution has been omitted, making the assay less hazardous. The assay is linear to 100 nmol Pi in the presence of 10mM ATP.