Purification and characterization of 3':5'-cyclic GMP-dependent protein kinase.

Abstract

Cyclic[c]GMP-dependent protein kinase was purified to homogeneity from bovine lung by affinity chromatography and characterized. Partially purified protein kinase, specifically activated by low concentrations of cGMP (22 nM), was adsorbed onto 8-(2-aminoethyl)-amino-cAMP-Sepharose. After washing to remove nonspecific proteins, cGMP-dependent protein kinase was specifically eluted by 0.1 mM cGMP. The purified protein contained cGMP-dependent protein kinase and specific cGMP binding activities. Purification of the holoenzyme was possible because subunit dissociation does not occur upon cyclic nucleotide binding. cGMP-dependent protein kinase holoenzyme has an apparent MW of 150,000 as determined by glycerol density gradient sedimentation. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, a single protein band of MW 74,000 was observed that suggested that holoenzyme is a dimer composed of subunits of identical molecular weight. cGMP-dependent protein kinase required high concentrations of Mg2+ for optimal activity; a heat-stable protein kinase modulator which inhibited cAMP-dependent protein kinase activity has no effect on the activity of purified cGMP-dependent protein kinase.