Kinases and ATPases produce adenosine diphosphate (ADP) as a common product, so an assay that detects ADP would provide a universal means for activity-based screening of enzymes in these families. Because it is known that most kinases accept ATPβS (sulfur on the β-phosphorous) as a substrate in place of adenosine triphosphate (ATP), the authors have developed a continuous assay using this substrate, with detection of the ADPβS product using dithio reagents. Such an assay is possible because dithio groups react selectively with ADPβS and not with ATPβS. Thiol detection was done using both Ellman’s reagent (DTNB) and a recently developed fluorescent dithio reagent, DSSA. Therefore, the assay can be run in both absorbance and fluorescence detection modes. The assay was used to perform steady-state kinetic analyses of both hexokinase and myosin ATPase. It was also used to demonstrate the diastereoselectivity of hexokinase (R) and myosin ATPase (S) for the isomers of ATPβS, consistent with previous results. When run in fluorescence mode using a plate reader, an average Z′ value of 0.54 was obtained, suggesting the assay is appropriate for high-throughput screening.
Knighton DR, Zheng JH, Ten Eyck LF, Ashford VA, Xuong NH, Taylor SS, et al: Crystal structure of the catalytic subunit of cyclic adenosine monophosphate-dependent protein kinase. Science1991;253: 407-414.
2.
Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S: The protein kinase complement of the human genome. Science2002;298: 1912-1934.
3.
Cohen P: The role of protein phosphorylation in human health and disease. The Sir Hans Krebs Medal Lecture. Eur J Biochem2001;268: 5001-5010.
4.
Valigra L: Varying approaches to inhibiting kinases. Drug Disc Develop2005;8: 51-55.
5.
Parker GJ, Law TL, Lenoch FJ, Bolger RE: Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays. J Biomol Screen2000;5: 77-88.
6.
Prokopowicz AS, Brown MP, Wildeson JM, Jakes S, Labadia ME: Fluorescent probes for use in protein kinase inhibitor binding competition assays for screening applications. PCT Int Appl2005;49-49.
7.
Shults MD, Imperiali B: Versatile fluorescence probes of protein kinase activity. J Am Chem Soc2003;125: 14248-14249.
8.
Armstrong C, Cohen P: Assaying protein kinase activity using a common seven-residue epitope substrate and phospho-specific antibodies. PCT Int Appl2003;48-48.
9.
Ruzzene M, Pinna LA: Assay of protein kinases and phosphatases using specific peptide substrates. In Hardie G (ed): Protein Phosphorylation: A Practical Approach. 2nd ed.Oxford (UK): Oxford University Press, 1999.
10.
Umezawa Y: Assay and screening methods for bioactive substances based on cellular signaling pathways. J Biotechnol2002;82: 357-370.
11.
Singh P, Lillywhite B, Bannaghan C, Broad P: Using IMAP technology to identify kinase inhibitors: comparison with a substrate depletion approach and analysis of the nature of false positives. Comb Chem High Throughput Screen2005;8: 319-325.
12.
Turek-Etienne TC, Kober TP, Stafford JM, Bryant RW: Development of a fluorescence polarization AKT serine/threonine kinase assay using an immobilized metal ion affinity-based technology. Assay Drug Develop Technol2003;1: 545-553.
13.
Singh P, Harden BJ, Lillywhite BJ, Broad PM: Identification of kinase inhibitors by an ATP depletion method. Assay Drug Develop Technol2004;2: 161-169.
14.
Koresawa M, Okabe T: High-throughput screening with quantitation of ATP consumption: a universal non-radioisotope, homogeneous assay for protein kinase. Assay Drug Develop Technol2004;2: 153-160.
15.
Badalassi F, Nguyen HK, Crotti P, Reymond J-L: A selective HIV-protease assay based on a chromogenic amino acid. Helv Chim Acta2002;85: 3090-3098.
16.
Klein G, Reymond JL: An enantioselective fluorometric assay for alcohol dehydrogenases using albumin catalysed β-elimination of umbelliferone. Bioorg Med Chem Lett1998;8: 1113-1116.
17.
Wahler D, Reymond J-L: The adrenalin test of enzymes. Angew Chem Intl Ed Engl2002;41: 1229-1232.
18.
Klein G, Reymond JL: An enantioselective fluorogenic assay of acetate hydrolysis for detecting lipase activity antibodies. Helv Chim Acta1999; 3: 400-408.
19.
Sevestre A, Helaine V, Guyot G, Martin C, Hecquet L: A fluorogenic assay for trans ketolase from S. cerevisiae. Tetrahedron Lett2003;44: 827-830.
Gutierrez MC, Sleegers A, Simpson HD, Alphand V, Furstoss R: The first fluorogenic assay for detecting a Baeyer-Villagerase activity in microbial cells. Org Biomol Chem2003;1: 3500-3506.
22.
Veldhuyzen WF, Nguyen Q, McMaster G, Lawrence DS: A light-activated probe of intracellular protein kinase activity. J Am Chem Soc2003; 125: 13358-13359.
23.
Simeonov A, Bi X, Nikiforov TT: Enzyme assays by fluorescence polarization in the presence of polyarginine: study of kinase, phosphatase and protease reactions. Anal Biochem2002;304: 193-199.
24.
Kupcho K, Somberg R, Bulleit B, Goueli SA: A homogeneous non radioactive high throughput fluorogenic protein kinase assay. Anal Biochem2003;317: 210-217.
25.
Martin K, Steinberg TH, Cooley LA, Gee KR, Beechem JM, Patton WF: Quantitative analysis of protein phosphorylation status and protein kinase activity in microarrays using a novel protein phosphorylation sensor dye. Proteomics2003;3: 1244-1255.
26.
Ojida A, Mito-Oka Y, Inoue M, Hamachi I: First artificial receptors and chemosensors toward phosphorylated peptides in aqueous solution. J Am Chem Soc2002;124: 6256-6258.
27.
Eckstein F, Goody RS: Synthesis and properties of diastereomers of adeno-sine 5′-(O-1-thiotriphosphate) and adenosine 5′-(O-2-thiotriphosphate). Biochemistry1976;15: 1685-1691.
28.
Silverstein RM: The determination of the molar extinction coefficient of reduced DTNB. Anal Biochem1975;63: 281-282.
29.
Pullela PK, Chiku T, Carvan MJ, Sem DS: Fluorescence-based detection of thiols in vitro and in vivo using dithio probes. Anal Biochem2006; 352: 265-273.
30.
Wilson K, Walker J: Principles and Techniques of Practical Biochemistry. 5th ed.Cambridge, UK: University of Cambridge Press, 2000.
31.
Pollard TD: Assays for myosin. Methods Enzymol (Part B)1982;85: 123-130.
32.
Chacko S, Conti MA, Aldestein RS: Effect of phosphorylation of smooth muscle myosin on actin activation and calcium ion regulation. Proc Natl Acad Sci USA1977;74: 129-133.
33.
Morris CA, Wells AL, Yang Z, Chen LQ, Baldacchino CV, Sweeney HLet al: Calcium functionally uncouples the heads of myosin VI. J Biol Chem2003;278: 23324-23330.
34.
Jaffe EK, Cohn M: Phosphorous-31 nuclear magnetic resonances of the thiophosphate analogues of adenine nucleotides: effects of pH and Mg2+ binding. Biochemistry1978;17: 652-657.
35.
Jaffe EK, Cohn M: Diastereomers of the nucleoside phosphothioates as probes of the structure of the metal nucleotide substrates and of the nucleotide binding site of yeast hexokinase. J Biol Chem1979;254: 10839-10845.
36.
Viola RE, Raushel FM, Rendina AR, Cleland WW: Substrate synergism and the kinetic mechanism of yeast hexokinase. Biochemistry1982; 21: 1295-1302.
37.
Zhang ZH, Chung TDY, Oldenburg KR: A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen1999;4: 67-73.
