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Molecular and Cellular Biochemistry

, Volume 19, Issue 1, pp 31–42 | Cite as

Reversible inhibition of cyclic AMP-dependent protein kinase by insulin

  • Ronald J. Walkenbach
  • Richard hazen
  • Joseph Larner
General Articles

Summary

Extracts of fasted rat diaphragms, previously treated with or without insulin were assayed for glycogen synthase, protein kinase and cyclic [3H]-AMP binding. Treatment with insulin produced an elevation in the % of glycogen synthase I and a concurrent decrease in cyclic AMP-dependent protein kinase activity and cyclic [3H]-AMP binding. Analysis of extracts by disc gel electrophoresis demonstrated the inhibition of cyclic [3H]-AMP binding to involve the Type I protein kinase holoenzyme. Inhibition of protein kinase activity was most apparent in the presence of 0.2 µM cyclic AMP, with enzymatic activity of the insulin-treated extracts typically 60–65% of control. Higher assay concentrations diminished the difference between control and insulin-treated extracts and concentrations greater than 20 µm abolished it.

The inhibition of cyclic AMP-dependent protein kinase activity after insulin was a transient and labile phenomenon. The effect was independent of ATP concentration in the assay, but was sensitive to the pH of tissue extraction, requiring a pH of 7.0 to 8.4 to be observed.

Insulin-mediated inhibition of protein kinase activity was reversed upon preincubation of extracts at 0–2°. Relatively concentrated homogenates (<4 µl buffer/mg tissue) yielded extracts which exhibited little or no inhibition of protein kinase activity compared to extracts prepared from more dilute (6–10 µl/mg) homogenates. A model for the inhibition of the cyclic-AMP dependent protein kinase by an insulin-generated inhibitor which becomes directly associated with the Type 1 holoenzyme is proposed.

Keywords

Protein Kinase Protein Kinase Activity Tissue Extraction Dependent Protein Reversible Inhibition 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Abbreviations

cyclic AMP (cAMP)

Adenosine 3′,5′-monophosphate

Tricine

N-Tris (Hydroxy-methyl) methyl glycine

G-6-P

glucose-6-phosphate

MES

2-[N-morpholino]ethane sulfonic acid

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Copyright information

© Dr. W. Junk b.v. Publishers 1978

Authors and Affiliations

  • Ronald J. Walkenbach
    • 1
  • Richard hazen
    • 1
  • Joseph Larner
    • 1
  1. 1.Department of PharmacologyUniversity of Virginia School of MedicineCharlottesville

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