Phosphorylation state of acetyl-coenzyme A carboxylase. I. Linear inverse relationship to activity ratios at different citrate concentrations.
Acetyl-CoA carboxylase and its associated kinase have been purified to homogeneity from rat liver and, together with the catalytic subunit of liver protein phosphatase, used to study the effect of phosphorylation on the carboxylase activity. Phosphatase increases the carboxylase activity, whereas the kinase decreases it. A linear inverse relationship (correlation coefficient = 0.98) exists between phosphate incorporated by the kinase and the specific activity. The kinetics of activation by citrate show an increased Ka and a decreased Vmax for carboxylase preparations with increasing levels of phosphate. On this basis an enzymic test was devised for phosphate incorporated by the kinase. Thus the ratio of activities at 0 and 2 mM citrate is inversely proportional to the phosphate incorporated (correlation coefficient = -0.95), with 0.8 mol of P incorporated per mol of subunit decreasing the activity ratio from 0.5 to 0. This activity ratio method has an inherent internal control which makes it suitable for determining the level of protein-bound phosphate affecting the carboxylase activity in crude tissue extracts, and hence it should be useful for physiological studies. Tryptic maps of carboxylase labeled with radioactive phosphate by the carboxylase kinase indicate that the slightly less than 1 mol of P/mol of subunit is distributed equally between two peptides, whereas cAMP-dependent protein kinase phosphorylates these two sites and a third which may not affect activity.
D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D002951:Citrates / Q000494:pharmacology*; D007700:Kinetics; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D010446:Peptide Fragments / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014357:Trypsin
D002951:Citrates; D010446:Peptide Fragments; D011494:Protein Kinases; C033288:(acetyl-CoA carboxylase) kinase; D014357:Trypsin; D008025:Ligases; D000103:Acetyl-CoA Carboxylase