Isolation of active cAMP dependent protein kinases from calf ovaries: gel electrophoresis vs. gel electrofocusing

Calf ovarian cAMP dependent Protein Kinase A super(1) was isolated by adsorption onto DEAE-cellulose, gel chromatography on agarose-polyacrylamide copolymer, electrophoresis in a 6% polyacrylamdie gel, 0.2% in Triton X-100, and DEAF-chromatograph. The yield was 3.3 mg, representing 22% of the starting material. Purification was 400-fold. The product appears homogeneous on gel electrophoresis at pH 10.2, but DEAF-chromatography, gel electrofocusing and gel electrophoresis at pH 8.5 and 7.5 reveal two charge isomeric forms of the enzyme. Preparative methods for the isolation of Protein Kinase B and cAMP Binding Protein A in homogenous form were also developed, using modifications of the above-states procedure.