Activation of cyclic AMP-dependent protein kinase by epinephrine in proliferating lymphoid cells

Abstract

Activation of the cyclic AMP-dependent protein kinase in intact lymphosarcoma cells can be promoted by epinephrine. The lymphosarcoma protein kinase is approximately 90% Isozyme I. Using the synthetic peptide PK-1 (LeuArgArgAlaSerLeuGly) as substrate for the kinase, the cyclic AMP-dependent protein kinase activity was 95% of the total protein phosphotransferase activity in the cell extract. In control cells the optimum extraction buffer for preventing enzyme subunit dissociation or reassociation contained buffer (2( N-morpholino)ethanesulfonic acid), EDTA, 2-mercaptoethanol, and charcoal. The absence of charcoal or the presence of 0.14 m KCl in the buffer promoted enzyme dissociation in the extract. The phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine had no effect. In extracts from epinephrine-treated cells or extracts to which purified catalytic subunit of the cyclic AMP-dependent protein kinase was added, recovery of the total protein kinase activity was 25% of that predicted in experiments with control cells. Recovery of enzyme activity increased to 80–95% of the predicted value when 0.14 m KCl was included in the extraction buffer. Methods involving a two-buffer extraction procedure are presented as the optimum protocol for determining in vivo activation of the cyclic AMP-dependent protein kinase, Isozyme I. Using these methods, epinephrine (1 μ m) dissociated the cyclic AMP-dependent protein kinase essentially 100% in intact lymphosarcoma cells. The dissociation was apparently maintained for up to 60 min. Approximately 10–15% of the dissociated enzyme may be specifically associated with particulate cell fractions. Collectively the data emphasize the experimental difficulty inherent in determination of the extent of in vivo dissociation of the cyclic AMP-dependent protein kinase.