Purification and some properties of a cyclic AMP-binding protein from human erythrocyte membranes.
A cyclic AMP-binding protein of human erythrocyte membranes was solubilized with 0.3% Triton X-100 in 10 mM Tris-HCl (pH 7.4) at 4 degrees C, and purified by DEAE-cellulose column chromatography and affinity chromatography on a cyclic AMP derivative-fixed Sepharose 4B column. The purified cyclic AMP-binding protein showed a single band (molecular weight: 49,000) on examination by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the band was specifically labeled with a photoaffinity analogue of cyclic AMP, 8-N3-cyclic [2-3H]AMP. This protein bound 1.6 mol of cyclic AMP per molecule with an association constant of 3.8 x 10(9) M-1 and the optimum pH for binding was 7.4. The protein inhibited the activity of a purified protein kinase from human erythrocyte membranes [Suzuki, K., Terao, T., & Osawa, T. (1981) J. Biochem. 89, 1--11], while cyclic AMP restored the enzymic activity. The amino acid composition of this protein was different from those of cytoplasmic cyclic AMP-binding proteins. These observations indicate that the cyclic AMP-binding protein purified in this work is the regulatory subunit of a membrane bound cyclic AMP-dependent protein kinase.
D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000097:blood*; D055598:Chemical Phenomena; D002621:Chemistry; D002373:Cyclic AMP Receptor Protein; D003956:Dialysis; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004910:Erythrocyte Membrane / Q000032:analysis*; D004912:Erythrocytes / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D011494:Protein Kinases / Q000502:physiology; D012967:Sodium Dodecyl Sulfate
D000596:Amino Acids; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D012967:Sodium Dodecyl Sulfate; D011494:Protein Kinases