Effect of epinephrine or cAMP on cAMP-bound protein kinase holoenzymes in rat heart.
Nonperfused or epinephrine-perfused rat hearts were used to examine the relative amounts of adenosine 3',5'-cyclic monophosphate (cAMP)-free and cAMP-bound holoenzymes of type II cAMP-dependent protein kinase (cAK). Crude tissue extracts of nonperfused hearts were chromatographed in the absence or presence of [3H]cAMP using DEAE-high-performance liquid chromatography. A partially resolved cAMP-free peak of cAK eluted at 0.17 M NaCl, and an asymmetric peak containing bound [3H]cAMP eluted at a slightly higher NaCl concentration. The first peak contained a tetrameric holoenzyme [2 regulatory (R) subunits and 2 catalytic (C) subunits]. From analysis of R-to-C ratios, the [3H]cAMP-bound peak contained a mixture of tetrameric and trimeric (R2C) forms. Both cAMP-free and cAMP-bound holoenzyme forms were virtually inactive without added cAMP under the conditions used. [3H]cAMP dissociation rate studies revealed that the bound cAMP in the peak fraction was equally distributed in the two different binding sites of the enzyme. Compared with the cAMP-free form, the cAMP-bound enzyme in the peak fraction exhibited enhanced binding in nonequilibrium [3H]cAMP binding assays. The cAMP-bound holoenzymes were estimated to represent at least 64% of the total type II cAK in control extracts, and the cAMP-free form was largely converted to the cAMP-bound forms by perfusing hearts with epinephrine.
Jiang
H
H
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615.
Corbin
J D
JD
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002852:Chromatography, Ion Exchange; D000242:Cyclic AMP / Q000378:metabolism / Q000494:pharmacology*; D004837:Epinephrine / Q000494:pharmacology*; D008297:Male; D009994:Osmolar Concentration; D010477:Perfusion; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D000242:Cyclic AMP; D011494:Protein Kinases; D004837:Epinephrine