Declercq P E, Falck J R, Kuwajima M, Tyminski H, Foster D W, McGarry J D
J Biol Chem. 1987 Jul 15;262(20):9812-21.
The effects of various inhibitors of carnitine palmitoyltransferase I were examined in mitochondria from rat liver and skeletal muscle. Three types of inhibitors were used: malonyl-CoA (reversible), tetradecylglycidyl-CoA and three of its analogues (irreversible), and 2-bromopalmitoyl-CoA (essentially irreversible when added with carnitine). Competitive binding studies between labeled and unlabeled ligands together with electrophoretic analysis of sodium dodecyl sulfate-solubilized membranes revealed that in mitochondria from both tissues all of the inhibitors interacted with a single protein. While the binding capacity for inhibitors was similar in liver and muscle (6-8 pmol/mg of mitochondrial protein) the proteins involved were of different monomeric size (Mr 94,000 and 86,000, respectively). Treatment of mitochondria with the detergent, octyl glucoside, yielded a soluble form of carnitine palmitoyltransferase and residual membranes that were devoid of enzyme activity. The solubilized enzyme displayed the same activity regardless of whether carnitine palmitoyltransferase I of the original mitochondria had first been exposed to an irreversible inhibitor or destroyed by chymotrypsin. It eluted as a single activity peak through four purification steps. The final product from both liver and muscle migrated as single band on sodium dodecyl sulfate-polyacrylamide electrophoresis with Mr of approximately 80,000. The data are consistent with the following model. The inhibitor binding protein is carnitine palmitoyltransferase I itself (as opposed to a regulatory subunit). The hepatic monomer is larger than the muscle enzyme. Each inhibitor interacts via its thioester group at the palmitoyl-CoA binding site of the enzyme but also at a second locus that is probably different for each agent and dictated by the chemical substituent on carbon 2. Disruption of the mitochondrial inner membrane by octyl glucoside causes inactivation of carnitine palmitoyltransferase I while releasing carnitine palmitoyltransferase II in active form. The latter is readily purified, is a smaller protein than carnitine palmitoyltransferase I, and has the same molecular weight in liver and muscle. It is insensitive to inhibitors where on or off the mitochondrial membrane.
在大鼠肝脏和骨骼肌的线粒体中检测了肉碱棕榈酰转移酶I的各种抑制剂的作用。使用了三种类型的抑制剂：丙二酰辅酶A（可逆）、十四烷基缩水甘油辅酶A及其三种类似物（不可逆），以及2-溴棕榈酰辅酶A（与肉碱一起添加时基本不可逆）。标记配体与未标记配体之间的竞争性结合研究以及十二烷基硫酸钠增溶膜的电泳分析表明，在两种组织的线粒体中，所有抑制剂均与单一蛋白质相互作用。虽然肝脏和肌肉中抑制剂的结合能力相似（6-8 pmol/mg线粒体蛋白），但所涉及的蛋白质单体大小不同（分别为94,000和86,000）。用去污剂辛基葡糖苷处理线粒体，产生了可溶形式的肉碱棕榈酰转移酶和缺乏酶活性的残留膜。无论原始线粒体的肉碱棕榈酰转移酶I是否首先暴露于不可逆抑制剂或被胰凝乳蛋白酶破坏，增溶后的酶都表现出相同的活性。经过四个纯化步骤，它以单一活性峰洗脱。来自肝脏和肌肉的最终产物在十二烷基硫酸钠-聚丙烯酰胺电泳上迁移为单一条带，分子量约为80,000。这些数据与以下模型一致。抑制剂结合蛋白是肉碱棕榈酰转移酶I本身（而不是调节亚基）。肝脏单体大于肌肉酶。每种抑制剂都通过其硫酯基团在酶的棕榈酰辅酶A结合位点相互作用，但也在第二个位点相互作用，每个试剂的第二个位点可能不同，由碳2上的化学取代基决定。辛基葡糖苷破坏线粒体内膜会导致肉碱棕榈酰转移酶I失活，同时释放出活性形式的肉碱棕榈酰转移酶II。后者易于纯化，是一种比肉碱棕榈酰转移酶I更小的蛋白质，在肝脏和肌肉中的分子量相同。它对线粒体内外膜上的抑制剂均不敏感。
