Kerner J, Zaluzec E, Gage D, Bieber L L
Department of Biochemistry, Michigan State University, East Lansing 48824.
J Biol Chem. 1994 Mar 18;269(11):8209-19.
A post 30,000 x g particulate fraction was isolated from rat heart. This mixed membrane fraction is enriched in a carnitine palmitoyltransferase which is sensitive to both malonyl-CoA and etomoxiryl-CoA at concentrations that inhibit the malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo/CPT-I) of intact mitochondria. Tritiated etomoxiryl-CoA labels two proteins with the same molecular weight as the labeled proteins from rat heart mitochondria. Malonyl-CoA-sensitive carnitine palmitoyltransferase in the particulate fraction is stable to freeze-thawing, and the activity is not latent. These data show that the carnitine palmitoyltransferase associated with this particle is CPTo/CPT-I. Positive Western blots were obtained, with the particle using anti-CPTi/CPT-II at a molecular weight identical with the CPT1/CPT-II purified from rat heart mitochondria. Catalytic activity was purified to near homogeneity in approximately 40% yield. The purified protein has a molecular weight identical with CPTi/CPT-II, it cross-reacts with antibody against CPTi/CPT-II, it is not inhibited by malonyl-CoA or etomoxiryl-CoA, and mass spectral analyses of the tryptic peptides give the same molecular masses as CPTi/CPT-II, and, when mixed with equal amounts of CPTi/CPT-II, one uniform spot is found by two-dimensional electrophoresis. These data indicate that the catalytic subunit of CPTo/CPT-I is the same as CPTi/CPT-II. The average inhibition of the CPT of frozen-thawed particles is 71% by 50 nM etomoxiryl-CoA and 62% by 50 nM malonyl-CoA. The inhibitor sensitivity, but not the catalytic activity, is lost by solubilization in 1% Triton X-114; removal of Triton X-114 using Extracti-Gel D restores etomoxiryl-CoA and malonyl-CoA sensitivity (both 50 nM) of CPT to an average of 77 and 48%, respectively. Consistent with previous reports, these results show that CPTo/CPT-I is NOT inactivated by detergents, rather detergents both desensitize it to malonyl-CoA and alter its Vmax. These data show the assumption that CPTo/CPT-I is inactivated by detergents is untenable.
从大鼠心脏中分离出30,000 x g离心后的微粒部分。这个混合膜部分富含一种肉碱棕榈酰转移酶，该酶对丙二酸单酰辅酶A和依托莫昔利辅酶A敏感，其浓度能抑制完整线粒体中对丙二酸单酰辅酶A敏感的肉碱棕榈酰转移酶（CPTo/CPT-I）。氚标记的依托莫昔利辅酶A标记了两种蛋白质，其分子量与大鼠心脏线粒体中标记的蛋白质相同。微粒部分中对丙二酸单酰辅酶A敏感的肉碱棕榈酰转移酶对冻融稳定，且活性无潜伏性。这些数据表明与该微粒相关的肉碱棕榈酰转移酶是CPTo/CPT-I。使用抗CPTi/CPT-II对微粒进行Western印迹检测呈阳性，其分子量与从大鼠心脏线粒体中纯化的CPT1/CPT-II相同。催化活性以约40%的产率纯化至接近均一性。纯化后的蛋白质分子量与CPTi/CPT-II相同，它与抗CPTi/CPT-II抗体发生交叉反应，不受丙二酸单酰辅酶A或依托莫昔利辅酶A抑制，胰蛋白酶肽段的质谱分析给出的分子量与CPTi/CPT-II相同，并且当与等量的CPTi/CPT-II混合时，二维电泳显示为一个均匀的斑点。这些数据表明CPTo/CPT-I的催化亚基与CPTi/CPT-II相同。冻融微粒的CPT平均被50 nM依托莫昔利辅酶A抑制71%，被50 nM丙二酸单酰辅酶A抑制62%。在1% Triton X-114中溶解会使抑制剂敏感性丧失，但催化活性不受影响；使用Extracti-Gel D去除Triton X-114后，CPT对依托莫昔利辅酶A和丙二酸单酰辅酶A（均为50 nM）的敏感性分别恢复至平均77%和48%。与先前的报道一致，这些结果表明CPTo/CPT-I不会被去污剂灭活，而是去污剂使其对丙二酸单酰辅酶A脱敏并改变其Vmax。这些数据表明CPTo/CPT-I被去污剂灭活的假设是站不住脚的。
