Weis B C, Esser V, Foster D W, McGarry J D
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1994 Jul 22;269(29):18712-5.
To begin to explore the basis for the tissue-specific expression of mitochondrial carnitine palmitoyltransferase I (CPT I), we focused on three rat tissues (liver, heart, and skeletal muscle) in which the enzyme was known to display very different properties. In Northern blot analysis, a cDNA probe corresponding to liver CPT I readily hybridized to a 4.5-kilobase species of mRNA in liver and heart, but not in skeletal muscle. Using the same probe to screen a neonatal rat heart cDNA library, a full-length clone, surprisingly having 100% sequence identity to the liver CPT I cDNA, was isolated. The paradox was resolved by two additional experiments. First, in Western blots of mitochondrial membranes, an antibody raised against liver CPT I recognized the 88-kDa protein in heart, as well as in liver, but not in skeletal muscle. Second, high specific activity [3H]deschloroetomoxir (a covalent ligand for CPT I) reacted with a single form of CPT I in liver (approximately 88 kDa) and skeletal muscle (approximately 82 kDa), while proteins of both sizes were labeled in the cardiac myocyte. Tritiated ligand binding to the two heart proteins was blocked by excess unlabeled malonyl-CoA. It is concluded that liver and skeletal muscle each contains a single and distinct isoform of CPT I with monomeric size of approximately 88 and 82 kDa, respectively. The heart contains a CPT I protein of approximately 82 kDa in size (probably identical to the skeletal muscle protein) but, importantly, also expresses the liver-type enzyme. The results likely explain why previous studies of heart CPT I yielded an apparent Km for carnitine and I50 value for malonyl-CoA inhibition that were intermediate between those of the liver and skeletal muscle enzymes.
为了开始探究线粒体肉碱棕榈酰转移酶I（CPT I）组织特异性表达的基础，我们聚焦于三种大鼠组织（肝脏、心脏和骨骼肌），已知该酶在这些组织中表现出非常不同的特性。在Northern印迹分析中，与肝脏CPT I对应的cDNA探针很容易与肝脏和心脏中一种4.5千碱基的mRNA杂交，但在骨骼肌中则不杂交。用同一探针筛选新生大鼠心脏cDNA文库，分离出一个全长克隆，令人惊讶的是，它与肝脏CPT I cDNA具有100%的序列同一性。通过另外两个实验解决了这一矛盾。首先，在线粒体膜的Western印迹中，针对肝脏CPT I产生的抗体识别心脏以及肝脏中的88 kDa蛋白，但不识别骨骼肌中的该蛋白。其次，高比活性的[3H]去氯乙莫昔芬（CPT I的共价配体）与肝脏中单一形式的CPT I（约88 kDa）和骨骼肌中单一形式的CPT I（约82 kDa）发生反应，而在心肌细胞中这两种大小的蛋白都被标记。氚标记配体与这两种心脏蛋白的结合被过量的未标记丙二酰辅酶A阻断。结论是肝脏和骨骼肌各自含有一种单一且独特的CPT I同工型，单体大小分别约为88 kDa和82 kDa。心脏含有一种大小约为82 kDa的CPT I蛋白（可能与骨骼肌蛋白相同），但重要的是，也表达肝脏型酶。这些结果可能解释了为什么先前对心脏CPT I的研究得出的肉碱表观Km值和丙二酰辅酶A抑制的I50值介于肝脏和骨骼肌酶的相应值之间。
