[1] Assays of protein kinase

doi:10.1016/0076-6879(83)99034-1

Methods in Enzymology

Volume 99, 1983, Pages 3-6

https://doi.org/10.1016/0076-6879(83)99034-1 Get rights and content

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This chapter discusses the assays of the protein kinase. The radioisotopic method for cAMP-dependent protein kinase can be used with tissue homogenates and purified enzyme. As crude homogenates contain ATPase activity and the heat-stable protein kinase inhibitor, activities obtained following diethylaminoethyl (DEAE)-cellulose chromatography are greater than that of the initial homogenate for brain, heart, skeletal muscle, and liver. The method is generally applicable for all acceptor proteins except those acidic proteins, which are not positively charged at pH 1.8. Small basic peptides, such as Ser-peptide can also be employed. The phosphoric acid procedure can be used to monitor autophosphorylation of the type II R subunit and to measure incorporation of labeled substances into the C subunit. The same stoichiometries are obtained with this procedure as with trichloroacetic acid precipitation. The spectrophotometric assay is valuable in enzyme kinetic studies as it is continuous. Most of the ATPase activity observed is Ser-peptide and cAMP-dependent. The radioisotopic assay, however, is much more rapid for processing the large numbers of fractions associated with enzyme purification.

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Citation Excerpt :
Crystal violet has a strong affinity to the external surface of the DNA double helix and can stain DNA by intercalation [34]. In the lactate dehydrogenase assay, lactate is oxidized to pyruvate at the same time NAD is reduced to NADH [35,36]. These assays are readily monitored using the absorbance of NADH at 340 nm and belongs to a class of enzyme assays that takes advantage of the chromophore of the NADH/NAD couple.
This review focuses on cytotoxic rhenium compounds in terms of IC₅₀ values, their mode of action in biological systems and their status in clinical trials. Biological studies of different rhenium compounds ordered by the oxidation state of rhenium are presented. Numerous rhenium complexes are reported, with the greatest number of compounds containing a Re(I)(CO)₃⁺ core. A wide range of complexes has been designed using a combination of organometallic ligands, N- or S-based ligands, peptides, multidentate ligands and oxo groups. Design concepts based on membrane permeability and lipophilicity, membrane receptor targets and specific enzyme targets are presented. The cytotoxicity parameter IC₅₀ is shown for organometallic compounds, coordination complexes, clusters and Re(oxo) complexes. In addition, a brief summary of in vivo studies is given. A further summary of rhenium compounds subjected to clinical trials is presented to provide information about the classes of rhenium compounds that have been tested in human beings and the approaches used in these studies. Moreover, the comparability of the IC₅₀ values among the cytotoxicity studies is critically assessed to provide the basis for a summary of the most potent rhenium compounds according to their reported IC₅₀ values for each type of cancer. The summary of the structures for the most cytotoxic complexes allows the identification of structural similarities and basic features that could lead to their cytotoxicity and might be useful for future investigations. Finally, the information from the analysis of the rhenium compounds subjected to cellular studies is compared to data on the rhenium compounds that have been involved in in vivo evaluation and clinical trials.

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[1] Assays of protein kinase

Publisher Summary

Anal. Biochem.

Anal. Biochem.

Anal. Biochem.