TY - JOUR
T1 - Activation of cyclic AMP-dependent protein kinase isoenzymes
T2 - Studies using specific antisera
AU - Erlichman, Jack
AU - Bloomgarden, David
AU - Sarkar, Dwijen
AU - Rubin, Charles S.
N1 - Funding Information:
ACKNOWLEDGMENTS We wish to thank Alfred Eng for expert technical assistance and Maureen Sacci for preparation of this manuscript. Supported by Grants AM-27736 and GM-22792 from the National Institutes of Health and NP-377 from the American Cancer Society. Jack Erlich-man is the recipient of a Sinsheimer Scholar Award.
PY - 1983/11
Y1 - 1983/11
N2 - A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nm for protein kinase I and 80 nm for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [γ-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.
AB - A novel method for rapidly determining the amount and degree of association-dissociation of the Type I and Type II cAMP-dependent protein kinases has been developed and validated. Antibodies directed against the regulatory subunits of Type I and Type II cAMP-dependent protein kinases were used. The antibodies formed complexes with holoenzymes and regulatory subunits which were precipitated by goat anti-rabbit IgG (immunoglobulin G). These complexes bound [3H]cAMP with an apparent Kb of 20 nm for protein kinase I and 80 nm for protein kinase II. Immunoprecipitated protein kinases I and II were catalytically active when incubated with cAMP, [γ-32P]ATP, and histone H2B. When mixtures of the two kinase isoenzymes or cytosol were incubated with various amounts of [3H]cAMP and the isoenzymes were separated by precipitation with antisera specific for each isoenzyme, the amount of [3H]cAMP associated with immunoprecipitates was proportional to the concentration of [3H]cAMP. In contrast, the catalytic activity that was immunoprecipitated varied inversely with the concentration of [3H]cAMP, showing that the activation of protein kinase could be assessed by the disappearance of catalytic activity from the immunoprecipitates. In the absence of MgATP protein kinase I was activated by a 10-fold lower concentration of cAMP than protein kinase II. However, when MgATP was added to the incubation, there was no significant difference in the binding of [3H]cAMP or dissociation of catalytic subunits of the two isoenzymes. The anti-R antibodies were also used to rapidly quantitate the concentration of regulatory subunits and the relative ratio of protein kinases I and II in tissue cytosols.
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U2 - 10.1016/0003-9861(83)90356-9
DO - 10.1016/0003-9861(83)90356-9
M3 - Article
C2 - 6605725
AN - SCOPUS:0021032589
SN - 0003-9861
VL - 227
SP - 136
EP - 146
JO - Archives of Biochemistry and Biophysics
JF - Archives of Biochemistry and Biophysics
IS - 1
ER -