Trumble G E, Smith M A, Winder W W
Department of Chemistry, Brigham Young University, Provo, Utah 84602, USA.
Eur J Biochem. 1995 Jul 1;231(1):192-8. doi: 10.1111/j.1432-1033.1995.tb20686.x.
An acetyl-CoA carboxylase has been purified from rat hindlimb muscle using ammonium sulfate fractionation and avidin-Sepharose affinity chromatography. SDS/PAGE of the isolated enzyme showed a major protein band at approximately 272 kDa and a minor band at 265 kDa. The liver acetyl-CoA carboxylase gave a major protein band at 265 kDa and a minor band at 280 kDa. Adipose tissue acetyl-CoA carboxylase migrated to the 265-kDa position on the gel. Western blots performed using streptavidin-alkaline-phosphatase suggest that the bands from the three tissues contain biotin. The present study has characterized the muscle and adipose tissue enzymes under steady-state kinetics and determined Michaelis constants for the substrates. The activation constant for citrate, an essential activator for both preparations, was 2.13 +/- 0.05 mM for the muscle enzyme and 3.02 +/- 0.12 mM for adipose tissue (P < 0.01). The Km values for the muscle acetyl-CoA carboxylase compared to the adipose tissue acetyl-CoA carboxylase were: ATP, 57.6 +/- 0.9 microM compared to 106.5 +/- 2.6 microM, P < 0.01; acetyl-CoA, 31.7 +/- 1.5 microM compared to 21.5 +/- 1.0 microM, P < 0.01; bicarbonate, 2.25 +/- 0.10 mM compared to 2.73 +/- 0.29 mM, P > 0.05. The muscle acetyl-CoA carboxylase was inhibited by malonyl-CoA (Ki = 10.6 +/- 1.0 microM) and palmitoyl-CoA (Ki = 2.2 +/- 0.3 microM). These properties are consistent with the hypothesis that regulation of acetyl-CoA carboxylase plays an important role in governing the rate of fatty acid oxidation in the skeletal muscle.
利用硫酸铵分级分离和抗生物素蛋白-琼脂糖亲和层析法，从大鼠后肢肌肉中纯化出了一种乙酰辅酶A羧化酶。对分离出的酶进行十二烷基硫酸钠/聚丙烯酰胺凝胶电泳（SDS/PAGE）分析，结果显示在约272 kDa处有一条主要蛋白带，在265 kDa处有一条次要蛋白带。肝脏中的乙酰辅酶A羧化酶在265 kDa处有一条主要蛋白带，在280 kDa处有一条次要蛋白带。脂肪组织中的乙酰辅酶A羧化酶在凝胶上迁移至265 kDa位置。使用链霉抗生物素蛋白-碱性磷酸酶进行的蛋白质免疫印迹分析表明，来自这三种组织的条带均含有生物素。本研究对肌肉和脂肪组织中的酶进行了稳态动力学特征分析，并测定了底物的米氏常数。柠檬酸盐是这两种制剂的必需激活剂，其对肌肉酶的激活常数为2.13±0.05 mM，对脂肪组织的激活常数为3.02±0.12 mM（P<0.01）。与脂肪组织中的乙酰辅酶A羧化酶相比，肌肉中的乙酰辅酶A羧化酶的米氏常数如下：ATP为57.6±0.9 μM，而脂肪组织中的为106.5±2.6 μM，P<0.01；乙酰辅酶A为31.7±1.5 μM，而脂肪组织中的为21.5±1.0 μM，P<0.01；碳酸氢盐为2.25±0.10 mM，而脂肪组织中的为2.73±0.29 mM，P>0.05。肌肉中的乙酰辅酶A羧化酶受到丙二酰辅酶A（抑制常数Ki = 10.6±1.0 μM）和棕榈酰辅酶A（抑制常数Ki = 2.2±0.3 μM）的抑制。这些特性与以下假设一致，即乙酰辅酶A羧化酶的调节在控制骨骼肌中脂肪酸氧化速率方面起着重要作用。
