Abstract
Heat-denatured chicken egg white lysozyme and the reduced carboxymethylated maleylated derivative of this protein were found to serve as substrates for rabbit skeletal muscle cyclic AMP-dependent protein kinase. The native form of the protein was not a substrate. Two phosphoryl groups per mole of lysozyme were incorporated in the reaction. It was determined that the phosphoryl moieties were bound to serine 24 and serine 50 in the modified protein. Serine 24 was phosphorylated approximately 3 times as fast as serine 50. Reduced carboxymethylated maleylated derivatives of bovine serum albumin, phosphorylase b, and creatine kinase also served as substrates for the protein kinase whereas their native forms did not. The reduced carboxymethylated maleylated derivative of the inhibitory subunit of troponin was a poorer substrate than the native form of the protein. Maleylated histones F1 and F2b were also poorer substrates than the nonderivatized forms. The significance of these experiments with reference to the specificity of cyclic AMP-dependent protein kinase is discussed.
Highlights
Reduced carboxymethylated maleylated derivatives of bovine serum albumin, phosphorylase b, and creatine kinase served as substrates for the protein kinase whereas their native forms did not
The protein was dissolved in 6 M urea and the amino groups were maleylated [21]. This protein, reduced carboxymethylated maleylated (RCMM-) lysozyme, was soluble under the conditions used for phosphorylation
Phosphorylation of Denatured Lysozyme-It was found that native lysozyme is not a substrate for the cyclic AMP-dependent protein kinase of rabbit skeletal muscle
Summary
Six times crystallized chicken egg white lysozyme was reduced with mercaptoethanol and S-carboxymethylated by standard procedures [20] and precipitated by dialysis against water. The protein was dissolved in 6 M urea (ultrapure from Schwarz/Mann) and the amino groups were maleylated [21] This protein, reduced carboxymethylated maleylated (RCMM-) lysozyme, was soluble under the conditions used for phosphorylation. This method was used for determining the maximal extent of 32P incorporation into proteins and in some experiments for rough estimates of reaction rates. In the latter case the time of incubation was adjusted so that the phosphorylation was still progressing at the time the reactions were terminated. Phosphorylated peptides were located on the paper by their radioactivity and by ninhydrin staining (2 g of ninhydrin, 100 ml of collidine, and 1900 ml of 95% ethanol) of guide strips, and were eluted with 30% acetic acid
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