In vitro phosphorylation and inactivation of rat liver acetyl-CoA carboxylase purified by avidin affinity chromatography.
Acetyl-CoA carboxylase (EC 6.4.1.2) has been isolated from rat liver by an avidin-affinity chromatography technique. This preparation has a specific activity of 1.17 +/- 0.06 U/mg and appears as a major (240,000 dalton) and minor (140,000 dalton) band on SDS-polyacrylamide gel electrophoresis. Enzyme isolated by this technique can incorporate 1.09 +/- 0.07 mol phosphate per mol enzyme (Mr = 480,000) when incubated with the catalytic subunit of the cyclic AMP-dependent protein kinase at 30 degrees C for 1 h. The associated activity loss under these conditions is 57 +/- 4.0% when the enzyme is assayed in the presence of 2.0 mM citrate. Less inactivation is observed when the enzyme is assayed in the presence of 5.0 mM citrate. The specific protein inhibitor of the cyclic AMP-dependent protein kinase blocks both the protein kinase stimulated phosphorylation and inactivation of acetyl-CoA carboxylase. The phosphorylated, inactivated rat liver carboxylase can be partially dephosphorylated and reactivated by incubation with a partially purified protein phosphatase. Preparations of acetyl-CoA carboxylase also contained an endogenous protein kinase(s) which incorporated 0.26 +/- 0.11 mol phosphate per mol carboxylase (Mr = 480,000) accompanied by a 26 +/- 9% decline in activity. We have additionally confirmed that the rat mammary gland enzyme, also isolated by avidin affinity chromatography, can be both phosphorylated and inactivated upon incubation with the cyclic AMP-dependent kinase.
D000103:Acetyl-CoA Carboxylase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D001360:Avidin; D002846:Chromatography, Affinity / Q000379:methods; D000242:Cyclic AMP / Q000378:metabolism; D008025:Ligases / Q000378:metabolism*; D008099:Liver / Q000201:enzymology*; D008297:Male; D008321:Mammary Glands, Animal / Q000201:enzymology; D008970:Molecular Weight; D010047:Ovalbumin / Q000031:analogs & derivatives; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D051381:Rats; D011919:Rats, Inbred Strains
D047428:Protein Kinase Inhibitors; D001360:Avidin; D010047:Ovalbumin; D000242:Cyclic AMP; D008025:Ligases; D000103:Acetyl-CoA Carboxylase