Regulation of Adenosine 3′,5′-Monophosphate-dependent Protein Kinase

Abstract In order to investigate the effects of hormones on the adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase of adipose tissue, it was necessary to study the characteristics of the enzyme in crude tissue extracts. There was an apparent activation of the enzyme by NaF, but this effect was probably due to inhibition of adenosine triphosphatase activity. The activity of phosphodiesterase in the extracts was low under the conditions of the experiments. Two procedures to determine the state of activation of the protein kinase in crude extracts are described. Both procedures are based on the amounts of inactive protein kinase (holoenzyme) and active catalytic subunit present. In the first procedure the activity ratio of the protein kinase is measured, i.e. the ratio of activity in the absence to activity in the presence of cAMP. At 0° in the presence of 0.5 m NaCl the protein kinase activity ratio was not affected by dilution of the extracts. Under these conditions, when cAMP was removed by gel filtration, the activity ratio decreased only very slowly but decreased rapidly in the absence of NaCl. The salt probably inhibits association of the regulatory and catalytic subunits of the protein kinase. In the second procedure for determining the activation state of the protein kinase, crude adipose tissue extracts were chromatographed on Sephadex G-100 columns and the areas under the curves of the two peaks of enzyme activity corresponding to holoenzyme and catalytic subunit were estimated. Sodium chloride (0.5 m) at 0° inhibited the association of the protein kinase subunits during this procedure. Sephadex G-100 profiles of the protein kinase in crude extracts of heart, brain, and liver were also determined. Sodium chloride (0.5 m) was also found to increase the activity ratio of the protein kinase in the presence of cAMP. At 0° this increase occurred relatively slowly and was accompanied by an increase in cAMP binding and a decrease in the cAMP requirement for enzyme activity.