title: New activity assays for ENPP1 with physiological substrates ATP and ADP.
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.
authors.affiliation: Conrad Prebys Center for Chemical Genomics, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA.
authors.forename: Chen-Ting
authors.initials: CT
authors.lastname: Ma
authors.forename: Eduard A
authors.initials: EA
authors.lastname: Sergienko
mesh_terms: D000244:Adenosine Diphosphate / Q000737:chemistry*; D000255:Adenosine Triphosphate / Q000737:chemistry*; D002460:Cell Line; D019902:Chondrocytes / Q000201:enzymology; D057075:Enzyme Assays; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D010006:Osteoblasts / Q000201:enzymology; D010710:Phosphates / Q000032:analysis; D010727:Phosphoric Diester Hydrolases / Q000032:analysis* / Q000737:chemistry; D011755:Pyrophosphatases / Q000032:analysis* / Q000737:chemistry
chemical_list: D010710:Phosphates; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010727:Phosphoric Diester Hydrolases; C065962:ectonucleotide pyrophosphatase phosphodiesterase 1; D011755:Pyrophosphatases