Winz R, Hess D, Aebersold R, Brownsey R W
Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, Canada.
J Biol Chem. 1994 May 20;269(20):14438-45.
Rat liver acetyl-CoA carboxylase (ACC, EC 6.4.1.2) exhibits major and minor subunits (M(r) of 265,000 and 280,000 respectively), the structure and function of which are compared in this study. The two subunits copurified and each contained biotin as demonstrated by avidin reactivity and direct determination of biocytin. In agreement with previous studies, the ACC subunits could be distinguished with specific monoclonal antibodies and differential tissue expression. We now report extensive differences in primary structure revealed by peptide mapping, mass spectrometric analysis of peptides following reverse phase high performance liquid chromatography, 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), 12 peptides derived from the 280-kDa subunit exhibited entirely novel sequences or matched partially (average 70% identity) with sequences within the 265-kDa subunit. The 280-kDa subunit may also exhibit distinct functional properties, since the initial rate of phosphorylation was at least 10-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. These structural studies suggest that the 265- and 280-kDa components (isozymes) of ACC are so distinct they may be encoded by separate genes, while the differential phosphorylation observed in vitro suggests a key role for the 280-kDa subunit in regulating enzyme activity within intact cells.
大鼠肝脏乙酰辅酶A羧化酶（ACC，EC 6.4.1.2）有大亚基和小亚基（相对分子质量分别为265,000和280,000），本研究对其结构和功能进行了比较。这两个亚基可共同纯化，且通过抗生物素蛋白反应性和生物胞素的直接测定表明每个亚基都含有生物素。与先前的研究一致，ACC亚基可通过特异性单克隆抗体和不同的组织表达来区分。我们现在报告通过肽图谱分析、反相高效液相色谱后肽的质谱分析以及选定肽的微测序所揭示的一级结构存在广泛差异。对源自265 kDa亚基的四个肽进行了测序，并与大鼠265 kDa ACC预测结构内的序列相匹配。虽然在两个亚基中检测到一个相同的肽序列（265 kDa亚基的2009 - 2024位残基），但源自280 kDa亚基的12个肽呈现出全新的序列，或与265 kDa亚基内的序列部分匹配（平均70%的同一性）。280 kDa亚基可能还具有独特的功能特性，因为在存在环磷酸腺苷依赖性蛋白激酶的情况下，其磷酸化的初始速率比265 kDa亚基至少高10倍。二维图谱显示从两个ACC亚基释放的胰蛋白酶磷酸肽是不同的。这些结构研究表明，ACC的265 kDa和280 kDa组分（同工酶）差异显著，可能由不同基因编码，而体外观察到的差异磷酸化表明280 kDa亚基在完整细胞内调节酶活性方面起关键作用。
