AJP - Heart and Circulatory Physiology, Vol 260, Issue 3 722-H729, Copyright © 1991 by American Physiological Society
Effect of epinephrine or cAMP on cAMP-bound protein kinase holoenzymes in rat heart
H. Jiang and J. D. Corbin
Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232-0615.
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.
