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Abstract

Acetyl—C0A carboxylase (ACC, EC 6.4.1.2) catalyzes the reaction which commits carbon to denovo synthesis of longchain fatty acids and is therefore a key regulated enzyme. ACC purified from rat white adipose tissue is a 265 kDa subunit; a 280 kDa subunit is the major, if not sole, isoform of ACC isolated from heart muscle. The rat liver enzyme exhibits major and minor subunits (Mr of 265,000 and 280,000 respectively), the structure and function of which are compared in this study. The two liver subunits co— purified and each contained biotin as demonstrated by avidin reactivity and direct determination of biocytin. The ACC subunits could be distinguished with specific monoclonal antibodies and differential tissue expression. Extensive differences in primary structure were revealed by peptide mapping, mass spectrometric analysis of peptides following reverse phase HPLC and microsequencing of selected peptides. Four peptides derived from the 265 kDa subunit were sequenced and matched sequences within the predicted structure of rat 265 kDa ACC. Although one identical peptide sequence was detected within both subunits (residues 2009-2024 of the 265 kDa subunit), twelve peptides derived from the 280 kDa subunit exhibited entirely novel sequences or matched partially (average 70% identity) with sequences within the 265 kDa subunit. In view of the established significance of reversible phosphorylation in the regulation of ACC, it was of interest to investigate the phosphorylation of the individual 280 and 265 kDa polypeptides. The 280 kDa subunit may also exhibit distinct functional properties, since the initial rate of phosphorylation was at least ten-fold greater than that of the 265 kDa subunit in the presence of cAMP-dependent protein kinase. Two-dimensional mapping demonstrated that the tryptic phosphopeptides released from the two ACC subunits are distinct. Similar results were also seen when the ACC subunits were phosphorylated with a MAP kinase or a CDC2 protein kinase. ACC prepared by avidin affinity chromatography contains a co-purifying kinase activity, in which MAP kinase, cAMP-dependent protein kinase, and AMPactivated protein kinase activities were all present. Preliminary evidence suggests that the 280 kDa subunit of ACC contains an epitope similar to the G-X—G-X-X-G motif conserved in protein kinase catalytic domains. The 265 and 280 kDa components (isozymes) of ACC are so distinct that they may be encoded by separate genes. Differential phosphorylation observed in vitro suggests a key role for the 280 kDa subunit in regulating enzyme activity within intact cells. In fact, when isolated cardiac myocytes were briefly exposed to the beta—adrenergic agonist,isoproterenol, 280-ACC, which is the major subunit expressed in this tissue, became rapidly phosphorylated. It is therefore probable that beta-adrenergic stimulation of PICA is directly responsible for the phosphorylation of 280- ACC and the inhibition of ACC activity. This is in marked contrast to 265-ACC, which does not appear to be a substrate for PKA in VIVO. It seems quite likely that in heart and skeletal muscle, a decrease in malonyl—C0A levels causes these cells to go from a state of carbohydrate catabolism, fatty acid esterification, malonyl—C0A production, and carnitine acyltransferase-l inhibition to a state of ACC inhibition and fatty acid oxidation. The phosphorylation of 280—ACC by PKA may control this change. In tissues such as liver where it is the quantitatively minor subunit, 280-ACC may still be involved in switching the metabolic focus from carbohydrate catabolism and fatty acid synthesis to fatty acid oxidation. Whatever the role in liver and muscle, it is intriguing that the 280 kDa subunit appears not to be expressed in adipose tissue, suggesting that the important sites of lipogenesis might exhibit distinctive control mechanisms.