Isolation of Active cAMP Dependent Protein Kinases from Calf Ovaries: Gel Electrcphoresis vs. Gel Electrofocusting

Abstract

Calf ovarian CAMP dependent protein kinase A was isolanted by adsorntion on to DEAF-cellulose, gel chromatography on agarosepolyacrylamide copolymer, electrophoresis in a 6% polyacrylamide gel, 0.2% in Iriton X-100, and DEAF-chromatography. The yield was 3.3 mg, representinw 22% of the starting material.

Purification was 400-fold. The product appears homogeneous on gel electropheresis at p 10.2, but DEAF-chromatography, gel elecectro focusing and gel electrophoresis at pH 8.5 and 7.5 reveal two charge isomeric forms of the enzyme.

Optimization of gel concentration for the separation of the enzyme from its closest migrating contaminant pointed to gel electrofocusing, rather than electrophoresis, as the appropriate Separation tool. However, that electrophoresis, as the inactivate the enzyme when conducted on wide-diameter preparative gels, if allowed to proceed to the steady-state, using either Ampholine or buffers as the carrier ampholytes, and etyleneglycol to repress no I yacrylami decopolyner, electrophorus is in a 6 po lyacrylaniide el, 0.7 in TM on -1 00, and DSAF-chromatography. The yield was 3.1 me, representing 787% of the starting material Petrification was 400-fold. The product appears homos jeneousonge I electrophoresis at pM 10.7, hut DEAF- chromatography, gel electron focus inn and ruels ectroohores is at pH 6.5 and 7.5 reveal two charred is on ericforirs of the enzyme, Opticalization of feel concentration for the separation of the enzyme from its closest ml floating cont eminent pointed to jel electro focusing, rather then elect rophoresis, as the appropriate separation tool. However, that method proved to inactivate the enzyme when c on ducted on wide-diameter preparative gels, if all owed to proceed to the steady-state, usinp; either Ampholine or buffers as the carrier ampholytes, and ethyl eneglycol to repress isoelectric precipitation. Only buffer electrofoucing on ultrogel Aca 54 if stoppert prior to the attainment of the isoelectric endpoint of the enzyme succeeded in recovering substantial (65%) activity, albeit at the price of resolution. Thus, a non-optimal concentration in polyacrylamide gel electrophoresis was applied in preference to preparative gel electro focusing.

Preparative methods for the isolation of Proter Kinase 8 and CAMP Binding Pritein A in homogeneous form were also developed, using nodifications of the above-stated procedure.