M. Castagna, W. K. Palmer, D. Walsh
May 1, 1977
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0
Influential Citations
15
Citations
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Journal
Archives of biochemistry and biophysics
Abstract
Abstract The perfusion of rat liver with N6,O2′-[3H]dibutyryl cyclic adenosine 3′,5′-monophosphate (Bt2-cAMP) has been used as a method to evaluate the site(s) of action of cyclic adenosine 3′,5′-monophosphate (cAMP). The metabolites produced from [3H]Bt2-cAMP have been determined by two-dimensional thin layer chromatography and electrophoresis. The accumulation of [3H]Bt2-cAMP within the cell was associated with the formation of N6-monobutyryl cyclic adenosine 3′,5′-monophosphate (N6-Bt-cAMP) and a product tentatively identified as either N6-monobutyryl AMP or N6,O2′-dibutyryl AMP. The latter product achieved a higher concentration in the liver than any other metabolite of [3H]Bt2-cAMP. Gel filtration of cytosol performed rapidly after termination of perfusion identified binding of [3H]N6-Bt-cAMP and [3H]cAMP to the macromolecular fraction. This binding was only decreased slightly if all extraction procedures were performed in the presence of cAMP, thus indicating that the binding observed occurred while the cells were intact. The only binding species detected in cytosol could be attributed to the regulatory subunit of the cAMP-dependent protein kinase. This assignment was made on the basis of: (i) sedimentation characteristics (5.2 S), (ii) the interaction with the catalytic subunit of skeletal muscle cAMP-dependent protein kinase, (iii) the equivalent degree of dependence of this interaction with the catalytic subunit on ATP-Mg2+ in correlation with the isozyme composition of the protein kinase in rat liver, (iv) the binding to nitrocellulose membranes, and (v) the correlation of the extent of binding of [3H]N6-Bt-cAMP and [3H]cAMP with the amount of 3H-labeled nucleotides produced within the cell and with the respective Ka values of the two cyclic nucleotides for the cAMP-dependent protein kinase. Treatment of the fraction with a 1:500 ratio of trypsin at 0 °C for 5 min results in the conversion of the binding species to a form that sediments at 3.2 S. A similar form was identified in extracts from frozen tissues, possibly due to the release of lysosomal proteases.