Cyclic GMP-activated protein kinase from Dictyostelium discoideum

Cells of Dictyostelium discoideum respond to their chemoattractants, cAMP and folate, with a rapid increase of the cellular cGMP content. The molecular mechanisms of cGMP action are not understood. Since in many biological systems cGMP-activated protein kinase is a prominent cGMP acceptor, we searched for such an enzyme in D. discoideum. By means of affinity chromatography on cGMP-Sepharose and other chromatographic procedures (DEAE-Trisacryl, CM-Trisacryl), we separated a novel protein kinase. This preparation did not show any regulation by cGMP and may represent an enzyme modified by proteolysis. In order to establish a rapid and efficient purification step, an antiserum against the kinase preparation was raised and coupled to Sepharose. Chromatography of the supernatant from a cell homogenate on this antibody matrix yielded a protein kinase that was activated 3-fold by cGMP. Half-maximal activation occurred at about 1 nM cGMP. Cyclic AMP at a 20-fold higher concentration also activated the protein kinase. On a Superose 6HR column the cGMP-activated protein kinase eluted in the same volume as enolase (Mr = 82,000).