The regulation of fatty acid biosynthesis: Simple procedure for the purification of acetyl CoA carboxylase from lactating rabbit mammary gland, and its phosphorylation by endogenous cyclic AMP-dependent and -independent protein kinase activities

Abstract

A number of key regulatory enzymes in eukaryotic cells are now known to be controlled by phosphorylation-dephosphorylation mechanisms, and there is increasing evidence that these interconversion reactions underlie the short term regulation of metabolism by external physiological stimuli (reviewed [I] ). The rate at which cytoplasmic acetyl CoA is converted to long chain fatty acids is stimulated by insulin in fat cells [2] and inhibited by glucagon in hepatocytes [3] . Acetyl CoA carboxylase has been implicated as the enzyme at which these hormonal effects are exerted, but the mechanisms are not understood in molecular terms. Acetyl CoA carboxylase is completely dependent on the allosteric activator citrate for activity, and it is inhibited by very low concentrations of palmityl CoA [4] . There is also preliminary evidence which indicates that acetyl CoA carboxylase may be regulated by a phosphorylation-dephosphorylation mechanism. Relatively crude preparations from rat liver [S] or bovine mammary gland [6] undergo a time-dependent inactivation if they are incubated with magnesium ions and [Y-~~P] ATP [7-91 and 32P-radioactivity is incorporated into a protein(s) which is precipitated by antiserum raised to a proteolytic fragment of rat liver acetyl CoA carboxylase [7-91 . Furthermore the inactivation can be reversed by incubation with a protein phosphatase preparation partially purified from hen oviduct [9] . If isolated fat cells are incubated with 32P, a protein with a subunit molecular weight corresponding to acetyl CoA carboxylase becomes labelled, and this protein can be precipitated using a monospecific antibody raised to acetyl CoA carboxylase from rat mammary gland [lo] . This experiment shows that the enzyme is phosphorylated in intact cells. We describe here an extremely simple method for the purification of large amounts of undergraded acetyl CoA carboxylase and demonstrate that this preparation can be phosphorylated by two distinct protein kinase activities that are present as trace endogenous contaminants in the preparation.