title: Characterization of the kinase domain of the ephrin-B3 receptor tyrosine kinase using a scintillation proximity assay.
abstract: We have characterized a recombinantly expressed N-terminally tagged GST fusion of the tyrosine kinase domain of human EphB3. The EphB3 kinase domain was shown to phosphorylate a group of synthetic tyrosine-containing peptides derived from a proprietary biotinylated kinase-biased peptide substrate library. In addition, the enzyme activity was stimulated by the divalent cation, manganese, and inhibited by addition of magnesium. The most active tyrosine-containing peptide, a biotinylated 49-mer, displayed saturation kinetics with an apparent K(m) of approximately 0.4 microM. The apparent K(m) for ATP was determined to be approximately 3 microM. The kinetics of the reaction was linear from concentrations of enzyme of 0.5 to 2 nM, and at or below the K(m) concentrations of the two substrates for at least 2 h at room temperature. Moreover, the tryrosine kinase inhibitor, PP2, produced an IC(50) of roughly 0.8 microM. In addition, the enzyme tolerated the solvent DMSO and was stable to multiple freeze/thaw cycles. Stability of the enzyme at 4 degrees C storage was seen out to 6 h with an approximately 50% reduction of activity by 24 h. Formatting the assay in a 384-well microtiter plate produced good uniformity of signal at 100% inhibition, 50% inhibition, and no inhibition. The coefficient of variance was at or below 10% with a signal-to-background ratio of approximately 24 and a z value of 0.72. Collectively, these results showed the ability to configure a robust HTS for a truncated recombinantly expressed family member of the Ephrin tyrosine kinases.
authors.affiliation: Millenium Pharmaceuticals, Inc., Cambridge, MA 02139, USA. bembenek@mpi.com
authors.forename: Michael E
authors.initials: ME
authors.lastname: Bembenek
authors.forename: Stephen
authors.initials: S
authors.lastname: Schmidt
authors.forename: Ping
authors.initials: P
authors.lastname: Li
authors.forename: Jennifer
authors.initials: J
authors.lastname: Morawiak
authors.forename: Andrea
authors.initials: A
authors.lastname: Prack
authors.forename: Sadhana
authors.initials: S
authors.lastname: Jain
authors.forename: Rebecca
authors.initials: R
authors.lastname: Roy
authors.forename: Thomas
authors.initials: T
authors.lastname: Parsons
authors.forename: Linda
authors.initials: L
authors.lastname: Chee
mesh_terms: D036389:Ephrin-B3 / Q000737:chemistry; D006801:Humans; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D036223:Receptor, EphB3 / Q000737:chemistry* / Q000235:genetics; D012588:Scintillation Counting / Q000379:methods*
chemical_list: D036389:Ephrin-B3; D011505:Protein-Tyrosine Kinases; D036223:Receptor, EphB3