Diurnal rhythm of phosphorylation of rat liver acetyl-CoA carboxylase by the AMP-activated protein kinase, demonstrated using freeze-clamping. Effects of high fat diets

Eur J Biochem. 1992 Feb 1;203(3):615-23. doi: 10.1111/j.1432-1033.1992.tb16591.x.


1. Acetyl-CoA carboxylase was purified to homogeneity, in the presence of protein phosphatase inhibitors, from rat liver sampled without freeze-clamping. The enzyme was in a highly phosphorylated state (4.8 mol/subunit) of low specific activity, and could be dramatically reactivated by treatment with protein phosphatase-2A. Amino acid sequencing and fast-atom-bombardment mass spectrometry showed that the enzyme was phosphorylated in Ser79, Ser1200 and Ser1215, the three sites known to be phosphorylated in cell-free assays by the AMP-activated protein kinase. 2. The inactive enzyme could also be completely reactivated using a limited treatment with trypsin, which removes the N-terminal segment containing Ser79 and reduces the phosphate content to 3.5 mol/subunit. These results strengthen previous findings that it is phosphorylation at Ser79 by the AMP-activated protein kinase that is responsible for the inactivation, and not the phosphorylation of the 220-kDa core fragment (which contains Ser1200 and Ser1215). 3. Analysis of the phosphorylation state of Ser79 in acetyl-CoA carboxylase from rat liver showed that phosphorylation occurs post mortem if freeze-clamping is not used. The higher phosphorylation observed in extracts made without freeze-clamping correlates with a large increase in AMP and decrease in ATP (presumably caused by hypoxia during removal of the liver), and with increased activity of the AMP-activated protein kinase. These results provide a rational explanation for the post mortem phosphorylation events, and re-emphasize the point that rapid cooling of cells and tissues is essential when measuring the expressed activity of acetyl-CoA carboxylase (as well as 3-hydroxy-3-methylglutaryl-CoA reductase). 4. Using the freeze-clamping procedure, the ratio of 'expressed' activity (measured in the presence of protein phosphatase inhibitors) to 'total' activity (measured after complete dephosphorylation) of rat liver acetyl-CoA carboxylase showed a marked diurnal rhythm, changing from 50% in the active form in the middle of the dark period to less than 10% active in the middle of the light period. The very low activity in the light period was associated with a high level of phosphorylation in Ser79. This diurnal rhythm is very similar to that previously described for the phosphorylation of 3-hydroxy-3-methylglutaryl-CoA reductase, another substrate for the AMP-activated protein kinase. Neither the activity of the AMP-activated protein kinase nor the content of AMP, ADP or ATP changed between the dark or light periods.(ABSTRACT TRUNCATED AT 400 WORDS)

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetyl-CoA Carboxylase / antagonists & inhibitors
  • Acetyl-CoA Carboxylase / isolation & purification
  • Acetyl-CoA Carboxylase / metabolism*
  • Adenine Nucleotides / metabolism
  • Adenosine Monophosphate / metabolism
  • Animals
  • Chromatography, High Pressure Liquid
  • Circadian Rhythm*
  • Dietary Fats / pharmacology*
  • Hydroxymethylglutaryl CoA Reductases / metabolism
  • Kinetics
  • Liver / enzymology*
  • Male
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Rats
  • Rats, Inbred Strains
  • Spectrometry, Mass, Fast Atom Bombardment
  • Substrate Specificity
  • Temperature


  • Adenine Nucleotides
  • Dietary Fats
  • Adenosine Monophosphate
  • Hydroxymethylglutaryl CoA Reductases
  • Protein Kinases
  • Acetyl-CoA Carboxylase