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  • Title: The use of affinity chromatography in purification of cyclic nucleotide receptor proteins.
    Author: Ramseyer J, Kanstein CB, Walton GM, Gill G.
    Journal: Biochim Biophys Acta; 1976 Oct 28; 446(2):358-70. PubMed ID: 186111.
    Abstract:
    Biospecific affinity chromatography has been used to purify specific cyclic AMP and cyclic GMP receptor proteins. Several variables are important for successful purification of the cyclic AMP receptor protein, the most critical being the length of the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to the aliphatic spacer side arm. 8-(2-Aminoethyl)-amino-cyclic AMP coupled to agarose specifically retains the cyclic AMP receptor protein by interaction with the immobilized nucleotide. Binding of the cyclic AMP receptor subunit of cyclic AMP-dependent protein kinase to the immobilized nucleotide results in dissociation of the catalytic protein phosphokinase subunit which is not retained. The retained cyclic AMP receptor protein is subsequently eluted by cyclic AMP. Homogeneous cyclic AMP receptor protein prepared from rabbit skeletal muscle by affinity chromatography has been characterized. The molecular weight of the native protein as determined by analytical ultracentrifugation and polyacrylamide gel electrophoresis at varying acrylamide concentrations is 76 800 and 82 000, respectively. The protein is asymmetric with frictional and axial ratios of 1.64 and 12. SDS and urea polyacrylamide gel electrophoresis indicate that the native cyclic AMP receptor is composed of two identical subunits of 42 700 molecular weight. The native protein dimer binds 2 moles of cyclic AMP per mole of protein and is active in suppressing activity of isolated catalytic subunits of cyclic AMP-dependent protein kinase. Cyclic GMP receptor protein from bovine lung has been purified using the same affinity chromatography media. Since cyclic nucleotide binding to cyclic GMP-dependent protein kinase does not result in dissociation of regulatory receptor and catalytic phosphotransferase subunits, the cyclic GMP-dependent protein kinase holoenzyme is retained on the column and can be subsequently specifically eluted with cyclic GMP.
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