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. Author manuscript; available in PMC: 2009 Sep 28.
Published in final edited form as: J Mol Biol. 2007 Aug 31;373(5):1281–1292. doi: 10.1016/j.jmb.2007.08.060

Figure 2. Kinetic parameters for Itk Y180F mutant and wild type Itk.

Figure 2

(A & B) The in vitro kinase activities of full-length Itk wild-type (WT), Y180F mutant and Y180F mutant in the presence of 2.5 μM free Itk SH2 domain are compared. Substrate (Peptide B or ATP) curves of WT full-length Itk (filled triangles), Itk Y180F (open squares), and Y180F mutant in the presence of free Itk SH2 domain (open circles) were fit to the Michealis-Menten equation using GraphFit to obtain the kinetic parameters reported in (B). (C) Western blot of purified Itk enzymes. 250 nM purified Itk WT or Y180F mutant enzymes were separated by SDS-PAGE, transferred onto PVDF, and probed with either an anti-pY180, anti-pY511 or anti-FLAG antibody. Y511 phosphorylation levels are comparable for WT and Y180F Itk. (D) Comparison of the activities of Itk full-length WT enzyme with that of the full-length kinase inactive (K390R) mutant. One μM enzyme was used for each reaction and Peptide B was used as substrate. Data shown are the average of at least two independent experiments.