Purification and characterization of two forms of cyclic GMP-dependent protein kinase from bovine aorta.
Cyclic GMP-dependent protein kinase in extracts of bovine aortic tissue eluted from DEAE-cellulose ion-exchange resins as two distinct peaks of activity. This elution pattern was preserved when the peaks were combined, precipitated with ammonium sulfate, dialyzed, and rechromatographed. Proteolysis did not appear to account for the two forms of kinase because (i) aging of the extract did not cause interconversion of the two forms, and (ii) both forms retained cGMP sensitivity unlike the proteolytically formed monomer. In addition, treatment with saturating concentrations of cGMP (10 microM) did not cause interconversion of the two forms. The first peak of cGMP-dependent protein kinase eluting from DEAE-cellulose (form 1) had a slightly greater mobility on gradient sodium dodecyl sulfate-polyacrylamide gels than the second peak (form 2). On native, nondenaturing gradient polyacrylamide gels, however, form 2 displayed the greater electrophoretic mobility. Furthermore, form 1, when bound to cAMP-agarose, appeared to exchange more rapidly with cGMP than form 2 when subjected to affinity chromatography. Peptide maps generated from the two forms by protease treatment were very similar, although trypsin produced a unique peptide in form 1 and Streptomyces griseus protease gave rise to unique peptides in forms 1 and 2. Phosphorylation did not appear to account for the physical differences because both enzymes could be phosphorylated to similar extents and dephosphorylation using alkaline phosphatase did not result in the conversion of one form to the other. These results suggest that either differences in primary structure or post-translational modification, other than phosphorylation, are responsible for the presence of two forms of cGMP-dependent protein kinase in aortic tissue.
Lincoln
T M
TM
Department of Pharmacology, College of Medicine, University of South Alabama, Mobile 36688.
Thompson
M
M
Cornwell
T L
TL
D000818:Animals; D001011:Aorta / Q000201:enzymology*; D002417:Cattle; D002846:Chromatography, Affinity; D002848:Chromatography, DEAE-Cellulose; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D010449:Peptide Mapping; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism
D011494:Protein Kinases