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J. Biochem, 1982, Vol. 92, No. 3 845-853
© 1982 Japanese Biochemical Society

research-article

Purification and Some Properties of a Cyclic AMP-Binding Protein from Human Erythrocyte Membranes

Kazuhiro SUZUKI*,1, Sumiko SUZUKI*, Tadao TERAO** and Toshiaki OSAWA*,2

*Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, The University of Tokyo Hongo, Bunkyo-ku, Tokyo 113
**Division of Radiochemistry, National Institute of Hygienic Sciences Setagaya-ku, Tokyo 158

2To whom all correspondence should be addressed

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°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 × 109 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.

1Present address: Division of Radiochemistry, National Institute of Hygienic Sciences, Setagaya-ku, Tokyo 158.


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