Declercq P E, Venincasa M D, Mills S E, Foster D W, McGarry J D
J Biol Chem. 1985 Oct 15;260(23):12516-22.
Malonyl-CoA and 2-tetradecylglycidyl-CoA (TG-CoA) are potent inhibitors of mitochondrial carnitine palmitoyltransferase I (EC 2.3.1.21). To gain insight into their mode of action, the effects of both agents on mitochondria from rat liver and skeletal muscle were examined before and after membrane disruption with octylglucoside or digitonin. Pretreatment of intact mitochondria with TG-CoA caused almost total suppression of carnitine palmitoyltransferase I, with concomitant loss in malonyl-CoA binding capacity. However, subsequent membrane solubilization with octylglucoside resulted in high and equal carnitine palmitoyltransferase activity from control and TG-CoA pretreated mitochondria; neither solubilized preparation showed sensitivity to malonyl-CoA or TG-CoA. Upon removal of the detergent by dialysis the bulk of carnitine palmitoyltransferase was reincorporated into membrane vesicles, but the reinserted enzyme remained insensitive to both inhibitors. Carnitine palmitoyltransferase containing vesicles failed to bind malonyl-CoA. With increasing concentrations of digitonin, release of carnitine palmitoyltransferase paralleled disruption of the inner mitochondrial membrane, as reflected by the appearance of matrix enzymes in the soluble fraction. The profile of enzyme release was identical in control and TG-CoA pretreated mitochondria even though carnitine palmitoyltransferase I had been initially suppressed in the latter. Similar results were obtained when animals were treated with 2-tetradecylglycidate prior to the preparation of liver mitochondria. We conclude that malonyl-CoA and TG-CoA interact reversibly and irreversibly, respectively, with a common site on the mitochondrial (inner) membrane and that occupancy of this site causes inhibition of carnitine palmitoyltransferase I, but not of carnitine palmitoyltransferase II. Assuming that octylglucoside and digitonin do not selectively inactivate carnitine palmitoyltransferase I, the data suggest that both malonyl-CoA and TG-CoA interact with a regulatory locus that is closely juxtaposed to but distinct from the active site of the membrane-bound enzyme.
丙二酰辅酶A和2-十四烷基缩水甘油辅酶A（TG-CoA）是线粒体肉碱棕榈酰转移酶I（EC 2.3.1.21）的强效抑制剂。为深入了解它们的作用方式，在用辛基葡糖苷或洋地黄皂苷破坏膜之前和之后，检测了这两种试剂对大鼠肝脏和骨骼肌线粒体的影响。用TG-CoA预处理完整的线粒体几乎完全抑制了肉碱棕榈酰转移酶I，同时丙二酰辅酶A结合能力丧失。然而，随后用辛基葡糖苷溶解膜后，对照线粒体和经TG-CoA预处理的线粒体产生了相同的高肉碱棕榈酰转移酶活性；两种溶解后的制剂对丙二酰辅酶A或TG-CoA均无敏感性。通过透析去除去污剂后，大部分肉碱棕榈酰转移酶重新整合到膜泡中，但重新插入的酶对两种抑制剂仍不敏感。含有肉碱棕榈酰转移酶的膜泡不能结合丙二酰辅酶A。随着洋地黄皂苷浓度的增加，肉碱棕榈酰转移酶的释放与线粒体内膜的破坏平行，这可通过可溶性部分中基质酶的出现来反映。尽管肉碱棕榈酰转移酶I在后者中最初受到抑制，但对照线粒体和经TG-CoA预处理的线粒体中酶的释放情况相同。在用2-十四烷基缩水甘油酸处理动物后制备肝脏线粒体时也得到了类似的结果。我们得出结论，丙二酰辅酶A和TG-CoA分别与线粒体（内）膜上的一个共同位点发生可逆和不可逆的相互作用，该位点的占据会导致肉碱棕榈酰转移酶I受到抑制，但不会抑制肉碱棕榈酰转移酶II。假设辛基葡糖苷和洋地黄皂苷不会选择性地使肉碱棕榈酰转移酶I失活，这些数据表明丙二酰辅酶A和TG-CoA都与一个调节位点相互作用，该位点与膜结合酶的活性位点紧密相邻但又不同。
