Purification and characterization of a catalytic subunit of an adenosine 3':5'-monophosphate-dependent protein kinase from human erythrocyte membranes.
Protein kinase [EC 2.7.1.37] of human erythrocyte membranes was solubilized with 0.5 M NaCl in 5 mM phosphate buffer, pH 6.7 at 4 degrees C and purified on a CM-Sephadex C-50 column, followed by affinity chromatography on a histone-Sepharose 4B column. The purified protein kinase gave a single band (molecular weight; 41,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 8.0 and a millimolar range of concentration of Mg2+ was required for its maximum activity. Histone and protamine were well phosphorylated by the protein kinase but casein and phosvitin were poor phosphate acceptors for the enzyme. The enzymic activity was not stimulated by cyclic AMP (cAMP). A cAMP-finding protein from human erythrocyte membranes inhibited the activity of the protein kinase, but the activity was restored with cAMP. A heat stable protein inhibitor from rabbit skeletal muscle also inhibited this enzyme. From these observations, this protein kinase seemed to be a catalytic subunit of the membrane bound cAMP-dependent protein kinase. This enzyme was strongly inhibited with Ca2+ in the presence of 1 mM MgCl2. Various sulfhydryl reagents and polyamines also had inhibitory activity on the protein kinase. Natural substrates of the enzyme were investigated using heat treated membranes and 0.5 M NaCl extracted membrane residues. Band 4.1, 4.2, and 4.5 proteins were phosphorylated but band 2 (spectrin) and band 3 proteins were poor substrates for this protein kinase.
D002352:Carrier Proteins; D002413:Cations, Divalent; D000242:Cyclic AMP / Q000494:pharmacology; D002373:Cyclic AMP Receptor Protein; D004910:Erythrocyte Membrane / Q000201:enzymology*; D004912:Erythrocytes / Q000201:enzymology*; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000097:blood; D010766:Phosphorylation; D011073:Polyamines / Q000494:pharmacology; D011494:Protein Kinases / Q000097:blood* / Q000302:isolation & purification; D013379:Substrate Specificity; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D002352:Carrier Proteins; D002413:Cations, Divalent; D002373:Cyclic AMP Receptor Protein; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011073:Polyamines; D013439:Sulfhydryl Reagents; D000242:Cyclic AMP; D011494:Protein Kinases