Measurement of acetyl-CoA carboxylase activity in isolated hepatocytes.
An assay is described for acetyl-CoA carboxylase activity in isolated hepatocytes. The assay is based on two principles: The hepatocytes are made permeable by digitonin. 64 micrograms of digitonin per mg of cellular protein were most effective in exposing enzyme activity without a significant effect on mitochondrial permeability. Enzyme activity is measured by coupling the carboxylase reaction to the fatty acid synthase reaction. The advantages offered by this procedure over existing assays are: rapidity, no need to prepare cell extracts, absence of product inhibition, no interference by mitochondrial enzymes, useful in systems with bicarbonate buffers, and simple separation of radioactive substrate from labelled products. Using this coupled enzyme assay a good correlation was observed between changes in the activity of acetyl-CoA carboxylase and changes in the rate of fatty acid synthesis in hepatocytes as effected by short-term modulators.
D000103:Acetyl-CoA Carboxylase / Q000032:analysis*; D000818:Animals; D002245:Carbon Dioxide; D004072:Digitonin; D064429:Fatty Acid Synthases; D008025:Ligases / Q000032:analysis*; D008099:Liver / Q000201:enzymology*; D008297:Male; D010539:Permeability; D051381:Rats; D011919:Rats, Inbred Strains
D002245:Carbon Dioxide; D064429:Fatty Acid Synthases; D008025:Ligases; D000103:Acetyl-CoA Carboxylase; D004072:Digitonin