title: Guanosine 3':5'-monophosphate-dependent protein kinase from bovine lung. Subunit structure and characterization of the purified enzyme.
abstract: cGMP-dependent protein kinase from bovine lung has been purified to homogeneity using 8-(2-aminoethyl)-amino adenosine 3':5'-monophosphate/Sepharose. Conditions for adsorption of holoenzyme to the affinity chromatography media followed by competitive ligand elution with cGMP have been determined. The holoenzyme of 150,000 molecular weight is composed of two 74,000 molecular weight subunits which are linked in part by disulfide bridges. Two moles of cGMP are bound per mol of holoenzyme compatible with 1 mol of cGMP/monomer. Dissociation of subunits does not occur upon cGMP binding and protein kinase activation. cGMP-dependent protein kinase has an isoelectric point of 5.4 and a Stokes radius of 50 A. The enzyme is asymmetric with an f/f0 of 1.42 and an axial ratio of 7.4. Determination of enzyme activity at varying concentrations of ATP revealed that cGMP increased the Vmax for ATP without significant effect on the Km. The purified enzyme was maximally active at 5 mM Mg2+; other divalent cations could not substitute for Mg2+. In the presence of Mg2+, strong inhibitory effects of other cations were observed with Mn2+, greater than Zn2+, greater than Co2+ greater than Ca2+. Although maximal cGMP-dependence was observed at pH 5.7 to 7.0, basal activity rose at higher pH values to approach activity observed with cGMP. A molecular model comparing cGMP-dependent protein kinase with cAMP-dependnet protein kinase is presented.
mesh_terms: D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D006152:Cyclic GMP / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D046911:Macromolecular Substances; D008274:Magnesium / Q000494:pharmacology; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D046911:Macromolecular Substances; D011494:Protein Kinases; D006152:Cyclic GMP; D008274:Magnesium