Abstract
Existing assays monitoring ENPP1 activity are either not physiologically relevant or not suitable for high-throughput screening (HTS). Here, we describe the development and implementation of two new ENPP1 activity assays that address these drawbacks. These assays employ physiological substrates of ENPP1, ATP and ADP. They rely on detection of inorganic phosphate using a special modification of the malachite green-molybdate colorimetric procedure that ensures stability of acid-labile compounds, such as the ones containing phosphodiester bonds. The pyrophosphate generated in ENPP1 reaction is converted to inorganic phosphate in the presence of inorganic phosphatase; whereas, omission of this coupling enzyme enables detection of the inorganic phosphate generated by ENPP1. These new ENPP1 assays were miniaturized into high-density microplate formats. With minimal requirement for ENPP1 enzyme, low micromolar phosphate detection sensitivity, and simple protocol involving three to four simple liquid handling steps, these robust assays are suitable for HTS.
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Acknowledgments
We are grateful to Dr. José Luis Millán and his lab for providing ENPP1 enzyme for these studies. This work is supported by NIH Roadmap grant # U54 HG005033 and Conrad Prebys Center for Chemical Genomics at Sanford-Burnham Medical Research Institute.
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Ma, CT., Sergienko, E.A. (2013). New Activity Assays for ENPP1 with Physiological Substrates ATP and ADP. In: Millán, J. (eds) Phosphatase Modulators. Methods in Molecular Biology, vol 1053. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-562-0_10
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DOI: https://doi.org/10.1007/978-1-62703-562-0_10
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Publisher Name: Humana Press, Totowa, NJ
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