Partial purification and characterization of an adenosine 3′,5′-monophosphate-dependent protein kinase from rabbit gastric mucosa

Abstract

An adenosine 3′,5′-monophosphate (cyclic AMP)-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) from rabbit gastric mucosa was partially purified and characterized. Purification by (NH 4) 2SO 4 precipitation, DEAE-cellulose chromatography, hydroxylapatite chromatography, and gel filtration increased the specific activity of the enzyme 60-fold. This protein kinase could be separated into a cyclic AMP-binding (regulatory) subunit and a catalytic subunit by chromatography on hydroxylapatite in the presence of cyclic AMP. The isolated catalytic subunit did not require cyclic AMP for activity. Recombination of the subunits in the presence of MgATP and bovine serum albumin restored cyclic AMP dependency to the catalytic subunit. Stokes radii of the catalytic subunit, the regulatory subunit, and the holoenzyme were found to be 2.6, 2.7 and 3.9 nm, respectively. The respective values for sedimentation coefficient were found to be 3.7, 3.5 and 5.1 S. Molecular weights calculated from these data gave values of 39 000 for both the catalytic and regulatory subunits and 82 000 for the holoenzyme. The enzyme phosphorylated histone, protamine, casein and endogeneous protein of the gastric mucosa. The apparent K m for ATP was 2·10 −5 M in the presence or absence of cyclic AMP. The concentration of cyclic AMP required for half maximal stimulation was 10 −8 M.