Enzyme assay protein kinase

Group II assays consist of those monitoring cellular second messengers. Thus, activation of receptors to cause Gs-protein activation of adenylate cyclase will lead to elevation of cytosolic or extracellularly secreted cyclic AMP. This second messenger phosphorylates numerous cyclic AMP-dependent protein kinases, which go on to phosphorylate metabolic enzymes and transport and regulatory proteins (see Chapter 2). Cyclic AMP can be detected either radiometrically or with fluorescent probe technology. 

To demonstrate the ability of the system to perform a matrix experiment as described above, concentrations of enzyme, substrate, and ATP were varied across the 24 wells in a row of an SBS 384-well microtiter plate. Results of these types of evaluations for the optimization of an assay for a protein kinase A and Kemptide system were presented by Wu et al.12 All the reactions were carried out in lOOmM HEPES, pH 7.4, lOmM MgCl2, lOmM DTT, and 0.015% Brij-35. No quenching agent was used. A sample from each of the 24 wells was analyzed in parallel every 6.5 min as the 24 enzymatic reactions progressed. 

Phosphorylation. To study regulation of enzyme activity, fractions were preincubated with 150 //M ATP, 5 U protein kinase catalytic subunit from bovine heart, 5 mM MgCl2, 60 mM dithiothreitol, 120 //M ATP, 5-80 fig protein depending on source and 50 mM Tris-HCl, pH 8. Samples were held 5 min at 35°C and assayed. 

In mammalian cells, there are multiple forms of enzymes within the same family, e.g., protein kinase C, phospholipase C, and caspase. A selective assay system would be instrumental in such cases to advance understanding of the respective role for each form within the same enzyme family. An example for design of a selective assay system for the superfamily of phospholipase A2 is provided in this chapter. Such a selective assay system may play a signihcant enabling role for PLA2 and other enzyme families in the discovery of inhibitors relevant to the treatment of pathologies involving those enzymes. 

A microchip-based assay for the enzymatic activity of protein kinase A (PKA) was conducted [1050]. A mixture of PKA (enzyme) and the fluorescently labeled substrate (kemptide, (LeuArgArgAlaSerLeuGly) was successively injected on a... 

Cohen, C.B., Chin-Dixon, E., Jeong, S., Nikiforov, T.T., A microchip-based enzyme assay for protein kinase A. Anal. Biochem. 1999, 273, 89-97. 

In contrast to enzyme assays, cell-based assays present the target in a more physiological milieu. With enzyme assays, it may be difficult to purify and express active kinases and phosphatases in their full-length forms and they may require the use of fusion proteins with kinase activity domains. Cell-based technologies, on the other hand, present the opportunity to express the targets with regulatory domains included. Furthermore, cell-based assays usually detect only cell-permeable inhibitors and have the potential to identify more unusual mechanisms, as described earlier. 

Myosin light chain kinase is a Ser/Thr-type protein kinase involved in regulation of smooth muscle. The enzyme is also found in smooth muscle and platelets. This assay uses a synthetic substrate that is not radioactively labeled. 

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