Regulation of rat liver acetyl CoA carboxylase
Creators: name: Quayle, Katherine Amanda
Publisher: name: University of British Columbia
Year: 2010

description: Acetyl-CoA carboxylase was purified 750-fold from rat liver cytosol using a modified purification procedure involving high speed ultracentrifugation, sucrose density step gradients, ion-exchange chromatography and avidin-sepharose affinity chromatography. The purified preparation had a specific activity of about 2 U/mg protein and a Km of 80uM for acetyl-CoA. Silver stain of the sample separated by denaturing polyacrylamide gel electrophoresis indicated 95% of the protein migrates as a single protein band with subunit Mr approximately 230Kd. There is approximately 2% contamination of low molecular mass material and 3-5% of the protein runs as a minor band of 240Kd. As the major protein band co-migrates on SDS-po1yacry1 amide gels with immunoprecipitated acetyl-CoA carboxylase from a fresh rat epididymal fat pad tissue extract, it appears that the 240Kd band in the rat liver sample may be a pre-form of the enzyme which undergoes post-translational modification to the mature form of the enzyme. Despite appearing almost homogeneous after silver-stain, the purified enzyme preparation still contains both cyclic AMP-independent and cyclic AMP-dependent protein kinase activity, the former causing incorporation of 0.1mol Pi/mol acetyl-CoA carboxylase subunit/hr, and the latter causing incorporation of 0.5mol Pi/mol subunit/hr. Addition of specific inhibitor to cyclic AMP-dependent protein kinase effectively blocks all cyclic AMP-dependent protein kinase activity. The cyclic AMP-independent protein kinase activity co-purifying with acetyl-CoA carboxylase phosphorylates the "control sites" as demonstrated by 2 -dimensional peptide analysis of radioactively labelled ACC. This phosphorylation has no apparent effect on enzyme activity. Phosphorylation by cyclic AMP-dependent protein kinase increases phosphorylation of the control sites and also results in incorporation of [32p] into the two A peptides and one of the B group of phosphopeptides. Neither of these endogenous protein kinase activities cause incorporation of the label into the insulin responsive site of acetyl-CoA carboxylase. At least 90% of the cyclic AMP-independent protein kinase activity (assayed using ACC as substrate) in the rat liver cytosol co-purifies with acetyl-CoA carboxylase upon sucrose density gradient centrifugation. Under the conditions described, approximately 90% of this protein kinase activity is separated from ACC by DEAE-ce11ulose chromatography, eluting with the other unbound protein from this column. 2-dimensiona1 tryptic peptide analysis of acetyl-CoA carboxylase phosphorylated by this protein kinase fraction increases incorporation of [32p] mainly into the control sites. Further fractionation of this sample however does not reveal increased I site phosphorylation. Protein kinase activity eluted together with ACC (at high salt) from DEAE-cellulose appeared to lead mainly to phosphorylation of the B group of peptides. However, in this case further fractionation of the DEAE-HS fraction on casein-sepharose produces a protein kinase fraction (eluted at 0.5M KC1) which brings about phosphorylation of the insulin-directed site of acetyl-CoA carboxylase. Thus a cyclic AMP-independent protein kinase activity which phosphorylates acetyl-CoA carboxylase at an insulin responsive site, has been partially purified from rat liver cytosol. We are therefore in a position to explore the effects of insulin-directed phosphorylation on the activity of acetyl-CoA carboxylase.