Purification and characterisation of the cyclic amp-dependent protein kinase, the C- and the R-protein from bovine liver

Abstract

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 ($\text{f}\text{f}{}_0$) 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 ($\text{f}\text{f}{}_0$ 1.1–1.26, axial ratio 3–5). The seondary structure with 35% α-helix, 19% β-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% α-helix, 21% β-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, A_280nm^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.