In-vitro kinase assay

Ponzetto, C., Wadewitz, A. G., Pendergast, A. M., Witte, O. N., and Wolgemuth, D. J. (1989). P I50c ai,/is detected in the mouse male germ line by an in vitro kinase assay and is associated in haploid cells with a stage-specific phosphoprotein. Oncogene 4 685-690. 

Immunoprecipitation, In Vitro Kinase Assays, and Western Blotting Clive Dickson... 

In vitro colorimetric assays were performed over a 1 pM to 100 (xM range in ligand concentration to assess the inhibition of phosphorylating activity by antibody recognition of phosphorylated peptide substrates [14]. The IC50 (50% inhibition concentration) for imatinib/Abl is 1 (xM, while the WBZ 4/Abl value is above 100 (xM (Fig. 8.7a). The active recombinant Abl kinase and its substrate (Abl-tide) were incubated in the presence of various WBZ 4 or imatinib concentrations and ATP (100 nM). Phosphorylation of Abl-tide peptide was detected by spectrophotometry following incubation with phospho-Abl-tide antibodies. 

Several homogeneous in vitro biochemical assays based on these systems have been described for proteases [139, 140], kinases [141, 142] [143], IL-2-IL-2R inter-... 

Portola Pharmaceuticals Inc, a US based biopharmaceutical company, are currently undertaking a Phase I study with an in-house oral Syk inhibitor PRT-062607 (structure unknown) to evaluate safety, PK and PD in healthy volunteers, ahead of investigating the drug in chronic inflammatory disease indications. Very scant information is known about the compound, except it is claimed to be highly selective for Syk from profiling in a broad panel of in vitro kinase and cellular assays. 

Chapter 1, this volume). Phosphorylation is also essential for movement of actin filaments in both the Nitella and the sliding actin in vitro motility assays (Umemoto and Sellers, 1990 Warshaw etai, 1990 Sellers etal., 1985). The site of the regulatory phosphorylation is Ser-19 on the LC20 (Pearson et al., 1984). Myosin light chain kinase (MLCK) also phosphorylates Thr-18, albeit at a much lower rate (Ikebe et al., 1986). Thr-18 phosphorylation increases the actin-activated MgATPase activity (Ikebe et al., 1988), but does not increase the rate of actin filament sliding in either of the two motility assays (Sellers et al., 1985 Okagaki et al, 1991). 

Fig. 3. In vitro Arf-GTPas assay to measure the GAP activity of ASAPl. (A) The stoichiometry of ASAPl tyrosine phosphorylation can be increased by in vitro kinase reaction performed with purified Flag-ASAP1/Pyk2 complexes. This increase is followed by Western blotting with an anti-phosphotyrosine antibody (pY) and levels of Hag-ASAPl are controlled with an anti-Flag antibody. (B) Arf-GTPase activities of nonphosphorylated (left panel) and phosphorylated ASAPl (right panel) are monitored in a fluorimetric Arfl-GTPase assay as a decrease of the intrinsic Arfl tryptophan fluorescence at 340 nm (in arbitrary units AU) upon excitation at 297.5 nm. Black dots indicate sample prior and grey dots after in vitro kinase reaction.

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