14] cGMP-dependent protein kinase activation in intact tissues

Abstract

Publisher Summary Unlike cAMP-kinase that dissociates into regulatory and catalytic subunits upon activation with cAMP, cGMP-kinase remains as a holoenzyme upon binding of cGMP. An increase in the activity of purified cGMP-kinase is observed after adding cGMP in sufficient amounts to occupy the cGMP-binding sites. In intact cells, the enzyme is probably activated in a similar manner. Ideally, when measuring the activation of cyclic nucleotide-dependent protein kinases in intact tissues, the equilibrium should not be disturbed by the procedures of tissue extraction or assaying. In the case of cAMP-kinase, the fact that the regulatory and catalytic subunits dissociate upon activation helps to preserve the activation state of the enzyme when it is diluted into homogenizing buffer and further diluted into a kinase reaction solution for assaying. In contrast, activated cGMP-kinase, under similar conditions, begins to lose activity, 4 probably because of the rapid dissociation of cGMP from the enzyme as a result of dilution. Therefore, special procedures are needed to minimize cGMP dissociation.