Notes: The authors of this review article highlight the use of bioluminescence as a readout for high-throughput ADME/Tox assays. They discuss three strategies for designing bioluminescent assays, using either luciferase, ATP or luciferin substrates as the limiting reagents for a luciferase-catalyzed reaction. Reporter gene assays limit the production of luciferase by tying it to a promoter or DNA regulatory region of interest. Such assays can be used to study genes that are regulated by drugs and other xenobiotics. Bioluminescent assays in which ATP is the limiting reagent of the luciferase reaction can be designed to monitor cell viability or the activity kinases. Bioluminescent assays in which the substrate is limiting can be designed so that the activity of a particular enzyme results in the production of a luciferin substrate that can, in turn, be acted upon by luciferase. (3926)

Notes: The authors of this study investigated the role of Lyn, a Src-family tyrosine kinase, in regulating the formation of the phagosome in alveolar macrophages in response to Psuedomonas aeruginosa (PA) infection. The Kinase-Glo^® Assay was used to assess Lyn activity, using acid-denatured enolase as the substrate. The authors found that Lyn kinase activity was increased following infection with PA. (3929)

Notes: The authors of this paper compared time-resolved, fluorescence energy transfer and ATP-based luminescent assays in ultrahigh-throughput screens (1536-well) for Rho-associated kinase II inhibitors. They found that both technologies are suitable for such a screen and perform similarly; however, they note that the ATP-based luminescent kinase assay provides an economical advantage. (3932)

Notes: Thees authors searched for small-molecule inhibitors of galactokinase (GALK). They developed an HTS assay using the Kinase-Glo^® Assay System. The HTS assay used 15 μM ATP and α-D-galactose as the substrate and was performed in 384-well plates against 50,000 small molecules. Two hundred compounds were identified from the primary screen as GALK inhibitors. (3934)

Notes: These authors used the Kinase-Glo^® Luminescent Kinase Assay to perform a high-throughput screening assay for inhibitors of glycogen synthase kinase-3β in 96-well plates. They used a 1µM ATP concentration and screened 55,000 compounds at 10µM. The assay sensitivity and IC₅₀ values of reference compounds were comparable to radioactive methods; the final optimized assay had an average Z´-factor of 0.72. (3591)

Notes: The authors of this paper review bioluminescent assay technologies, discussing HTS reporter, cell-based and luciferase biosensor assays. They divide luminescent assays into three basic categories: assays that measure ATP concentration (cell viability and kinase assays), assays that measure changes in luciferase levels (reporter assays, GPCR assays), and assays that measure changes in luciferin levels (protease [including caspase], P450 and MAO assays). (3737)

Notes: The authors developed a high-throughput assay in a 1536-well format using the Kinase-Glo^® Luminescent Kinase Assay to test for small-molecule ceramide kinase (CERK) inhibitors. The assay was performed using a final compound concentration of 10µM, and an ATP concentration of 5µM in a total volume of 5µl. The assay was robust with Z´-factors of 0.54. (3590)

Notes: The authors of this study investigated the role of okadaic acid (OA), a phosphatase inhibitor, in the regulation of the cell cycle using Vicia faba (fava bean) meristem tissue. They used the Kinase-Glo^® Luminescent Kinase Assay to analyze the activity of Histone H1 kinase in OA-treated and untreated meristem. Twenty micromolar histone was used as the substrate and the ATP concentration was 16µM for the assays. (3930)

Notes: The authors of this study investigated the effect of oxygen (hypoxia and normoxia) on endothelial nitric oxide synthase (NOS-3) and caveolin-1 functions in pulmonary microvascular endothelial cells (MVECs) isolated from fetal and neonatal lambs. To determine if protein kinase C (PKC) is involved in NOS-3 and caveolin-1 function, MVECs were cultured under hypoxic and normoxic conditions, and nuclear and cytosolic cell fractions were prepared. PKC activity was analyzed using the Kinase-Glo^® Plus Luminescent Kinase Assay with and without PKC-specific substrate. The reaction mixture contained 7.5mM ATP, 20mM MgCl, 5µg/ml substrate peptide and 50mM Tris. Reactions were incubated for 10 minutes. (3545)

Notes: The Kinase-Glo^® Luminescent Kinase Assay was used in 384-well plates to test a library of 720 peptides, potential substrates for protein kinase A. In addition, various concentrations of kinase 1 and 3 were tested in 384-well and 1536-well plates, and the results determined using the Kinase-Glo^® Assay. (3728)

Notes: This study investigated whether prequalene diphosphate (PSDP) is an endogenous regulator of phosphatidyl 3-kinase (PI3K). The Kinase-Glo^® Luminescent Kinase Assay was used to determine the activity of recombinant human p110gamma-PI3K activity. A total of 4pmol of PI3K/reaction was exposed to PDSP or a PI3K inhibitor followed by 25µg L-α-phosphatidylinositol and 1µM ATP before adding the Kinase-Glo^® Reagent. (3546)

Notes: The Kinase-Glo^® Luminescent Kinase Assay was used to screen inhibitors of glycogen synthase kinase-3 beta (GSK-3β). Reactions were performed in the presence of 25µM substrate, 1µM ATP, 50 mM Hepes, (pH 7.5), 1mM EDTA, 1mM EGTA, and 15mM magnesium acetate. Purified glycogen synthase kinase-3β was obtained from Upstate (catalog# 14-306). The substrate used in the assays was a synthesized prephosphorylated polypeptide. Inhibitors in DMSO vehicle were added to the reactions so that the final DMSO concentration did not exceed 1%. Reactions were incubated at 30°C for 30 minutes. Results were read on a FLUOstar POLARstar Optima (BMG Labtech) multimode reader. (3387)

Notes: Through immunoprecipitation studies it was found that levels of the proto-oncogenic serine/threonine kinase Pim-1 are regulated by ubiquitination. The kinase was found to associate with the chaperone proteins Hsp70 and Hsp90. Hsp70 associated with the ubiquitinated form of Pim-1, and Hsp90 protected Pim-1 from degradation. To study the activity of Pim-1 in these associations with chaperone proteins, recombinant Pim-1 protein was mixed with a five-fold excess of recombinant Hsp70 or Hsp90, or left unbound in the presence of a binding buffer [10 mmol/L HEPES (pH 7.4), 100 mmol/L KCl, 5 mmol/L DTT, 20 mmol/L Na2MoO₄, 50 mmol/L ATP]. These complexes were then immunoprecipitated with a Pim-1 monoclonal antibody and washed four times in kinase buffer [25 mmol/L HEPES, 10 mmol/L MgCl₂, 0.5 μg/mL DTT]. Finally, 0.1μmol/L ATP and 400 μmol/L of peptide substrate were added and the reaction incubated for 5 minutes at 30° C. The Kinase-Glo^® Luminescent Kinase Assay was used to determine the activity of Pim-1 in its various bound states. It was found that all binding configurations of the kinase were active, underscoring the importance of efficient degradation of the kinase as a means for regulating its activity. (3264)

Notes: The Kinase-Glo^® assay was used to assess Cdk4 kinase activity in HeLa cell lysates. In these experiments, 100μg of lysate (by protein determination) were immunoprecipitated using 1μg of a mouse monoclonal anti-Cdk4 antibody and 20μl protein G sepharose beads. The immobilized Cdk4 kinase immunoprecipitates were then washed three times in lysis buffer and twice in kinase buffer (50mM, pH 7.5, 10mM MgCl₂, 250μM EGTA, 10mM β-glycerophosphate, 100μM Na₃VO₄, 1mM DTT), and resuspended in 30μl of kinase buffer. Cdk4 Kinase activity was then assessed by incubating the immunoprecipitates with 20μl of 0.1μM ATP and 2μg Rb peptide for 30 minutes at 30°C. After this incubation the Kinase-Glo^® Detection Reagent was added and the resulting luminescence recorded. Data are presented as relative luminescence from various samples. (3304)

Notes: These authors investigated the mechanisms by which anti-carcinogenic compounds derived from Japanese and subtropical vegetables and fruits attenuate LPS-induced COX-2 mRNA expression in RAW264.7 mouse macrophages. Using Western blot analysis, 10µg nuclear or 20µg cytosolic protein fractions isolated from LPS-treated macrophages in the absence or presence of various phytochemicals were stained with several antibodies for signal pathway proteins, including the Anti-ERK1/2 pAb. Further analysis of the MAPK and NF-κB systems was performed using firefly luciferase constructs co-transfected with the control pRL-TK Vector at a 1:1 ratio. Transfected cells were exposed to the plant compound for 12 hours and then to LPS for a further 12 hours. Reporter activity was measured using the Dual-Luciferase^® Reporter Assay System. To determine the effect of the phytochemical zerumbone on the kinase reaction, a cell-free kinase assay was performed using Kinase-Glo^® Assay System. In this assay, 1.5µl recombinant MAPKAPK-2, 0.2µl recombinant active p38α, 1µl zerumbone and 4µmol/l ATP were incubated for 3 hours at 28°C before addition of an equal volume of Kinase-Glo^® Reagent. (3333)

Notes: The authors describe the advantages of the Kinase-Glo™ kit for high throughput screening. Cyclin-dependent kinase 4 (Cdk4) was used as a model kinase to draw comparisons between the Kinase-Glo™ assay and a “gold standard” radioactive filter assay in terms of reproducibility and use in compound inhibition studies. There is excellent description of the buffers, equipment and conditions used in the study. (3058)

Notes: In this paper,  lipopolysaccharide (LPS) induced PKC activity in human monocytic cell (U937) extracts was measured with the Kinase-Glo^® Luminescent Kinase Assay.  1 x 10⁷ U937 cells were cultured in the presence or absence of 5 μg/ml LPS for 4 hours.  The cells were then washed in PBS and resuspended in a kinase/extraction buffer (40 mM Tris [pH 7.5], 20 mM MgCl₂, 0.1 mg/ml BSA) before sonication. These extracts were then diluted in a PKC reaction buffer (20 mM Tris [pH 7.5], 10 mM MgCl₂, 0.1 mg/ml BSA, 250μM EGTA, 400μM CaCl₂, 0.32mg/ml phosphatidylserine, 0.032mg/ml diacylglycerol) with 10μM ATP and 100μM neurogranin_(28–43).  Results were displayed as relative light units compared to a control.  The researchers also mention normalizing the results by the Bradford protein determination assay. (3069)

Notes: The authors studied the structure and function of Protein Kinase A (PKA) as a model for protein kinases. Two amino acids with contacts to other side chains, Thr197 and Ser338, were replaced with Alanine, expressed and purified from E. coli, and the enzymatic activity compared to that of wildtype PKA. The Kinase-Glo^® Luminescent Kinase Assay and the Kemptide peptide were used to determine the activities of the wildtype and mutant PKAs in the presence and absence of the known inhibitor, H7. (3290)