Purification and characterisation of the cyclic AMP-dependent protein kinase, the C- and the R-protein from bovine liver.
A cyclic AMP-dependent protein kinase, its regulatory (R) and catalytic (C) protein were isolated from bovine liver, The cyclic AMP-dependent protein kinase showed two protein bands on SDS-polyacrylamide gel electrophoresis with molecular weights of 54 000 and 40 000. They correspond to the data for the separately isolated R-and C-protein. The molecular weight of the holoenzyme ranged from 172 000-179 000, depending on the estimation method. The molecular weight of the R-protein ranged from 97 000-98 000. This, and the results of the SDS-polyacrylamide gel electrophoresis, demonstrates a dimeric structure. The frictional ratios (f/fo) of 1.67-1.7 for the holoenzyme and 1.9 for the R-protein correspond to highly asymmetric shapes. Assuming a prolate form, the axial ratios are 13-14 and 17, respectively. The C-protein is globular (f/fo 1.1-1.26, axial ratio 3-5). The secondary structure with 35% alpha-helix, 19% beta-sheet and 49% aperiodic form of the holoenzyme is similar to the R-protein with 35, 19 and 46%, respectively. The C-protein contains 29% alpha-helix, 21% beta-sheet and 50% aperiodic form. The dimeric R-protein binds 4 mol cyclic AMP and can be phosphorylated in the presence of the C-protein. An absorption coefficient, A280nm 1.0%, of 5.4 was calculated for the R-protein and of 13.6 for C-protein. The data for C-protein, e.g., molecular weight, heterogeneity in isoelectrofocusing, phosphate content, etc., are in good agreement with those found by Sudgen, P.H. and Corbin, J.D. (1976) biochem. J. 159, 423-437.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D000242:Cyclic AMP / Q000494:pharmacology*; D004355:Drug Stability; D007525:Isoelectric Focusing; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D010710:Phosphates / Q000032:analysis; D011487:Protein Conformation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D000596:Amino Acids; D046911:Macromolecular Substances; D010710:Phosphates; D000242:Cyclic AMP; D011494:Protein Kinases