Mechanism of Action of Gonadotropin. III. Binding of Adenosine 3′,5′-Monophosphate by the 105,000 × g Supernatant Fraction from Homogenates of Calf Ovaries1

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Abstract
The binding of adenosine 3′,5′-monophosphate (cyclic AMP) to calf ovary cytosol has been investigated by equilibrium dialysis, membrane filter assay, and sucrose density gradient analysis. Protein purified from calf ovary cytosol bound cyclic AMP with an apparent dissociation constant kd of 4.1 × 10-9M. The cyclic AMP-binding protein complex exhibited a sedimentation coefficient of approximately 4S. The cyclic AMP-binding activity was inactivated by pronase, heat treatment, and by the addition of 0.1% SDS. The specificity of binding for cyclic AMP was tested by the addition of a variety of nucleotides to the reaction mixture and their ability to compete with cyclic [3H]AMP for the protein binding sites. At a concentration of 5 × 10-7M, only cyclic AMP competed effectively with cyclic [3H]AMP. At higher concentrations (2.5 × 10-5M), dibutyryl cyclic AMP, cyclic GMP, cyclic CMP, and cyclic UMP were able to displace cyclic [3H]AMP from the binding protein. Purification of the cyclic AMP-binding protein by DEAE-cellulose chromatography in the absence of added cyclic AMP resulted in the isolation of a cyclic AMP-dependent protein kinase fraction which contained cyclic AMP-binding activity. Protein kinase activity separated from the cyclic AMP-binding activity by subsequent polyacrylamide gel electrophoresis. Purification of the cytosol cyclic AMP-binding protein by DEAEcellulose chromatography in the presence of 5 nM cyclic AMP resulted in the resolution and recovery of two protein kinases, I and II. Cyclic AMP-independent protein kinase II exhibited no cyclic AMP-binding activity. Protein kinase I was associated with the majority of the cyclic AMP-binding activity. Polyacrylamide gel electrophoresis dissociated protein kinase I into a fraction exhibiting only cyclic AMP-binding activity and into a fraction with cyclic AMP-independent protein kinase activity. (Endocrinology93: 217,1973)