Hormonal regulation of cyclic-AMP-dependent protein kinase of rat diaphragm by epinephrine and insulin.

Abstract

1 The properties of protein kinase in extracts of rat diaphragm has been studied. Investigations on protein kinase has also been made after preincubation of the diaphragms with epinephrine or insulin. Protein kinase activity was measured by the rate of incorporation of 32P from [γ-32P]ATP into histone in the absence and the presence of cyclic AMP. In addition the binding of cyclic [3H]AMP by the extracts was investigated. Furthermore, the cyclic-AMP-dependent and -independent enzymes were separated by gel filtration on Sephadex G-150. 2 Protein kinase activity in crude extracts was increased 2- to 3-fold by cyclic AMP. The cyclic-AMP-dependent activity was relatively stable during purification with ammonium sulphate and recovered to an extent of 70%. The cyclic-AMP-dependent activity in crude extracts was recovered as a separate peak by gel filtration on Sephadex G-150. Purified cyclic-AMP-dependent protein kinase was quantitatively recovered when added to a crude muscle extract. It is indicated that the cyclic-AMP-dependent protein kinase in muscle extracts quantitatively reflects the amount of the inactive holoenzyme in the tissue. 3 Protein kinase activity independent of cyclic AMP was inactivated by crude muscle extracts. This was demonstrated by preincubation of muscle extracts with cyclic AMP. Dissociation of the holoenzyme by cyclic AMP as demonstrated by a decrease of cyclic-AMP-dependent activity and a decreased binding of cyclic [3H]AMP was not accompanied by a change in the cyclic-AMP-independent activity. Purified cyclic-AMP-independent protein kinase added to crude muscle extracts was also inactivated. A small peak of cyclic-AMP-independent activity was obtained when crude extracts were subjected to gel filtration on Sephadex G-150. This peak was increased 2- to 3-fold when elution was done in the presence of 0.5 M NaCl. It is indicated that the active catalytic subunit of protein kinase in the tissue mainly is converted to an inactive complex in muscle extracts. 4 After preincubation of rat diaphragms with epinephrine the cyclic-AMP-dependent activity as well as the binding of cyclic [3H]AMP were decreased. This was demonstrated both in crude muscle extracts and in purified enzyme preparations of protein kinase from the extracts. 5 After preincubation of rat diaphragms with insulin the opposite effects could be demonstrated. Both in crude extracts and purified enzyme preparations the binding of cyclic [3H]AMP and the cyclic-AMP-dependent protein kinase activity were increased. 6 By gel filtration of rat diaphragm extracts on Sephadex G-150 it was demonstrated that preincubation of the diaphragms with epinephrine resulted in decreased cyclic-AMP-dependent protein kinase, whilst the cyclic-AMP-independent enzyme was increased. The opposite effects were seen by preincubation of the diaphragms with insulin, i.e. increased cyclic-AMP-dependent and decreased cyclic-AMP-independent enzymes. 7 It is indicated that important metabolic effects by epinephrine and insulin are mediated by modulation of the activity of protein kinase in the tissue. The effect by epinephrine on protein kinase is attributed to the increase of cyclic AMP concentration. The mechanism of the action of insulin on this enzyme is discussed.