Lewin T M, Kim J H, Granger D A, Vance J E, Coleman R A
Departments of Nutrition and Pediatrics, University of North Carolina, Chapel Hill, NC 27599, USA.
J Biol Chem. 2001 Jul 6;276(27):24674-9. doi: 10.1074/jbc.M102036200. Epub 2001 Apr 23.
Inhibition studies have suggested that acyl-CoA synthetase (ACS, EC ) isoforms might regulate the use of acyl-CoAs by different metabolic pathways. In order to determine whether the subcellular locations differed for each of the three ACSs present in liver and whether these isoforms were regulated independently, non-cross-reacting peptide antibodies were raised against ACS1, ACS4, and ACS5. ACS1 was identified in endoplasmic reticulum, mitochondria-associated membrane (MAM), and cytosol, but not in mitochondria. ACS4 was present primarily in MAM, and the 76-kDa ACS5 protein was located in mitochondrial membrane. Consistent with these locations, N-ethylmaleimide, an inhibitor of ACS4, inhibited ACS activity 47% in MAM and 28% in endoplasmic reticulum. Troglitazone, a second ACS4 inhibitor, inhibited ACS activity <10% in microsomes and mitochondria and 45% in MAM. Triacsin C, a competitive inhibitor of both ACS1 and ACS4, inhibited ACS activity similarly in endoplasmic reticulum, MAM, and mitochondria, suggesting that a hitherto unidentified triacsin-sensitive ACS is present in mitochondria. ACS1, ACS4, and ACS5 were regulated independently by fasting and re-feeding. Fasting rats for 48 h resulted in a decrease in ACS4 protein, and an increase in ACS5. Re-feeding normal chow or a high sucrose diet for 24 h after a 48-h fast increased both ACS1 and ACS4 protein expression 1.5-2.0-fold, consistent with inhibition studies. These results suggest that ACS1 and ACS4 may be linked to triacylglycerol synthesis. Taken together, the data suggest that acyl-CoAs may be functionally channeled to specific metabolic pathways through different ACS isoforms in unique subcellular locations.
抑制研究表明，酰基辅酶A合成酶（ACS，EC ）同工型可能通过不同的代谢途径调节酰基辅酶A的利用。为了确定肝脏中存在的三种ACS各自的亚细胞定位是否不同，以及这些同工型是否独立调节，制备了针对ACS1、ACS4和ACS5的非交叉反应性肽抗体。在内质网、线粒体相关膜（MAM）和胞质溶胶中鉴定出ACS1，但线粒体中未鉴定出。ACS4主要存在于MAM中，76 kDa的ACS5蛋白位于线粒体膜中。与这些定位一致，ACS4的抑制剂N-乙基马来酰亚胺在MAM中抑制ACS活性47%，在内质网中抑制28%。另一种ACS4抑制剂曲格列酮在微粒体和线粒体中抑制ACS活性<10%，在MAM中抑制45%。ACS1和ACS4的竞争性抑制剂三辛素C在内质网、MAM和线粒体中对ACS活性的抑制作用相似，表明线粒体中存在一种迄今未鉴定的对三辛素敏感的ACS。ACS1、ACS4和ACS5通过禁食和重新喂食独立调节。禁食48小时的大鼠导致ACS4蛋白减少，ACS5增加。在禁食48小时后重新喂食普通饲料或高蔗糖饲料24小时，ACS1和ACS4蛋白表达均增加1.5 - 2.0倍，与抑制研究结果一致。这些结果表明ACS1和ACS4可能与三酰甘油合成有关。综上所述，数据表明酰基辅酶A可能通过独特亚细胞定位中的不同ACS同工型在功能上被导向特定的代谢途径。
