Activation of cyclic AMP-dependent protein kinase isoenzymes

Studies using specific antisera

Jack Erlichman, David Bloomgarden, Dwijen Sarkar, Charles S. Rubin

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)136-146
Number of pages11
JournalArchives of Biochemistry and Biophysics
Volume227
Issue number1
DOIs
StatePublished - 1983

Fingerprint

Cyclic AMP-Dependent Protein Kinases
Protein Kinases
Isoenzymes
Immune Sera
Chemical activation
Adenosine Triphosphate
Cytosol
Antibodies
Catalyst activity
Holoenzymes
Goats
Histones
Anti-Idiotypic Antibodies
Catalytic Domain
Phosphotransferases
Immunoglobulin G
Association reactions
Tissue
Rabbits

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Activation of cyclic AMP-dependent protein kinase isoenzymes : Studies using specific antisera. / Erlichman, Jack; Bloomgarden, David; Sarkar, Dwijen; Rubin, Charles S.

In: Archives of Biochemistry and Biophysics, Vol. 227, No. 1, 1983, p. 136-146.

Research output: Contribution to journalArticle

Erlichman, Jack ; Bloomgarden, David ; Sarkar, Dwijen ; Rubin, Charles S. / Activation of cyclic AMP-dependent protein kinase isoenzymes : Studies using specific antisera. In: Archives of Biochemistry and Biophysics. 1983 ; Vol. 227, No. 1. pp. 136-146.
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