Standardization of the assay for the catalytic subunit of cyclic AMP-dependent protein kinase using a synthetic peptide substrate.
Optimal assay conditions for analyses of the catalytic subunit activity of the cyclic AMP-dependent protein kinase using a well-defined, commercially available synthetic peptide as the phosphate acceptor are defined. Activity of purified catalytic subunit toward the synthetic peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (PK-1; Kemptide) was 1.5- to 45-fold greater than activity toward other commonly used substrates such as histone fractions, casein, and protamine. The effects of buffer, pH, Mg2+, and protein kinase concentration on activity toward PK-1 were investigated. The optimal assay conditions determined were as follows: 20 mM Hepes or phosphate buffer, pH 7.5, 100 microM PK-1, 100 microM [gamma-32P]ATP, 3 mM MgCl2, 12 mM KCl, and 20-200 ng of catalytic subunit assayed at 30 degrees C. Since PK-1 is the only commercially available, well-defined substrate for this enzyme, adaption of the proposed standard assay conditions for the analyses of purified catalytic subunit activity will permit direct comparison of kinetic parameters and purity of enzyme preparations from multiple preparations.
D000818:Animals; D002021:Buffers; D002352:Carrier Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D047908:Intracellular Signaling Peptides and Proteins; D008274:Magnesium; D015636:Magnesium Chloride; D008722:Methods; D009132:Muscles / Q000201:enzymology*; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D011817:Rabbits; D013379:Substrate Specificity
D002021:Buffers; D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; C015482:protein kinase modulator; D015636:Magnesium Chloride; D000242:Cyclic AMP; D011494:Protein Kinases; D008274:Magnesium