Ramsay R R, Derrick J P, Friend A S, Tubbs P K
Department of Biochemistry and Biophysics, University of California, San Francisco 94143.
Biochem J. 1987 Jun 1;244(2):271-8. doi: 10.1042/bj2440271.
A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.
一种新的肉碱棕榈酰转移酶（CPT）从牛肝线粒体中纯化至同质，根据过氧化氢酶和谷氨酸脱氢酶活性判断，该线粒体过氧化物酶体污染率为96%。该酶无需使用去污剂即可轻松从线粒体中去除。与其他线粒体CPT制剂不同，它是单体（分子量63,500），对肉豆蔻酰辅酶A和棕榈酰辅酶A活性最高。对于从己酰辅酶A到硬脂酰辅酶A的一系列底物，Km值在0.8至4微摩尔之间；这些值远低于其他纯化的CPT制剂报道的值。用棕榈酰辅酶A测定时，L-肉碱的Km为185微摩尔，且随链长变化不大。这也低于其他CPT制剂报道的值，但高于中链转移酶引用的值。动力学和抑制剂研究与快速平衡随机顺序机制一致。2-溴棕榈酰辅酶A是外膜CPT的抑制剂，在未预孵育的情况下加入时，以棕榈酰辅酶A作为可变底物竞争性抑制该酶。如果该酶与2-溴棕榈酰辅酶A和肉碱预孵育，蛋白质经凝胶过滤后活性不再恢复。抑制剂以每酶亚基1:1的化学计量比结合。
