EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF THE CGMP-DEPENDENT PROTEIN-KINASE-I-BETA AND PROTEIN-KINASE-II USING THE BACULOVIRUS SYSTEM

Detailed studies of differences in distinct cGMP kinase isoforms are h ighly dependent on expression of large amounts of these enzyme isoform s that are not easily purified by conventional methods, Here cGMP-depe ndent protein kinases, the type I beta soluble form from human placent a, and the type II membrane-associated form from rat intestine, were e ach expressed in a baculovirus/Sf9 cell system and purified in milligr am amounts by affinity chromatography. The expressed recombinant prote ins displayed characteristics like those of their native counterparts, cGK I beta was expressed as a 76 kDa protein predominantly found in t he cytosol fraction, whereas cGK II was expressed as an 86 kDa protein predominantly associated with the membrane fraction. The apparent K-a and V-max of cGMP for activation of cGK I beta were 0.5 mu M and 3.4 mu mol/min/mg, and for cGK II were 0.04 mu M and 1.8 mu mol/min/mg.