Methods in Enzymology Vol.99 Hormone Action, Part F: Protein Kinases

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.

• Series: Methods in Enzymology (Book 99)
• Hardcover: 480 pages
• Publisher: Academic Press; 3rd edition (October 12, 1983)
• Language: English
• ISBN-10: 012181999X
• ISBN-13: 978-0121819996
• Product Dimensions: 6 x 1 x 9 inches

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