Hormonal effects on fat cell adenosine 3′,5′-monophosphate dependent protein kinase

Abstract

Fat cell extracts were electrophoresed on polyacrylamide gels to separate the regulatory subunit and holoenzyme species of protein kinase. Gels were incubated with cyclic [ 3H]AMP ([ 3H]cAMP) and washed, and the bound [ 3H]cAMP was estimated. The band of [ 3H]cAMP found closest to the origin (Peak I) was associated with cAMP-dependent protamine kinase activity. A seond [ 3H]cAMP peak (Peak II) also contained protamine kinase activity. Although the kinase activity of Peak II was much less than Peak I, more [ 3H]-cAMP was bound in Peak II than in Peak I. The [ 3H]cAMP peak furthest from the origin (Peak III) was devoid of kinase activity. Incubation of extracts with cAMP prior to electrophoresis diminished or abolished kinase activity in Peaks I and II. This incubation also decreased [ 3H]cAMP binding in Peaks I and II, and increased binding in Peak III. When extracts were incubated with [ 3H]cAMP before electrophoresis, essentially all of the radioactivity was found in Peak III. It was concluded that Peak I represents a holoenzyme form and that Peak III is composed of the regulatory subunits of this enzyme. Peak II may represent a relatively inactive holoenzyme form not previously described. Incubation of adipocytes with epinephrine resulted in a dose- and time-dependent decrease in Peak I and increase in Peak III, and insulin opposed these effects of epinephrine. After 1-min incubations with epinephrine, the decreases in Peak I or increases in Peak III correlated with increases in phosphorylase a activity, decreases in glycogen synthase I activity and changes in cAMP, both in the presence and absence of insulin. However, after incubation with epinephrine for more than 2 min in the presence of insulin, phosphorylase a activity did not correlate with cAMP, suggesting that factors other than the cyclic nucleotide mediate the effects of epinephrine and insulin.