[26] Purification of type Iα and Type Iβ isozymes and proteolyzed type Iβ monomeric enzyme of cGMP-dependent protein kinase from bovine aorta

Publisher Summary This chapter describes the purification of bovine aorta type I α , type 1 β , and type 1 β monomer. The cGMP-dependent protein kinase activity is determined and the incorporation of [ 32 P]P i from [ γ - 32 P] adenosine triphosphate (ATP) into a synthetic heptapeptide (Arg-Lys-Arg-Ser-Arg-Ala-Glu) is used in the assay as this peptide is a relatively more specific substrate for the cGMP-dependent protein kinase than for the cAMP-dependent protein kinase. The typical assay is conducted using 50 μ l of an assay mixture which contains 20 m M Tris, pH 7.4, 200 μ M ATP, 136 μ g/ml of the heptapeptide, 20 m M MgCl 2 , 100 μ M 3-isobutyl-l-methylxanthine, 1 μM synthetic peptide inhibitor of the cAMP-dependent protein kinase, and 30,000 cpm/ μ l [γ- 32 P]ATP. The reaction is conducted in absence and presence of 10 μM cGMP in order to determine cGMP-dependent kinase activity. A 10 μ l aliquot of enzyme is added to the 50 μ l of assay mixture, and the reaction is allowed to proceed at 30° for 10 min. To terminate the reaction, a 50 μ l aliquot is removed from each reaction tube and then spotted onto individual phosphocellulose papers. The phosphocellulose papers are immediately dropped into 75 m M phosphoric acid (10 ml/paper), washed with four changes of phosphoric acid to remove unreacted [ γ - 32 P]ATP, and once with ethanol. The papers are then dried using a hair dryer and placed into vials containing scintillation fluid and counted. One unit of enzyme activity is defined as 1 μ mol of phosphate transferred from ATP to the peptide substrate per minute per milligram of enzyme.