Esser V, Britton C H, Weis B C, Foster D W, McGarry J D
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
J Biol Chem. 1993 Mar 15;268(8):5817-22.
We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase I (CPT I). Oligonucleotides corresponding to two tryptic peptides derived from the malonyl-CoA/etomoxir-CoA-binding protein of rat liver mitochondria (Esser, V., Kuwajima, M., Britton, C. H., Krishnan, K., Foster, D. W., and McGarry, J. D. (1993) J. Biol. Chem. 268, 5810-5816) were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone obtained consisted of a 102-nucleotide 5'-untranslated region, a single open reading frame of 2,319 bases predicting a protein of 773 amino acids (M(r) = 88,150), and a 3'-untranslated segment of 1,957 nucleotides followed by the poly(A)+ tail. A 0.9-kilobase fragment of the cDNA recognized a single species of mRNA (approximately 4.7 kilobases in size) in rat liver. The identity of the cDNA was confirmed by the findings that (i) the open reading frame encoded all four peptides found in the original protein; (ii) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV6, resulted in a selective and 10-20-fold induction of a malonyl-CoA- and etomoxir-CoA-sensitive CPT activity; and (iii) the overexpressed product was readily detected on Western blots by an antibody raised against the starting material. It seems likely that the de novo synthesized enzyme is targeted to the mitochondrial outer membrane via a leader peptide and that the mature protein achieves membrane anchoring through a stretch of 20 amino acids present near its amino terminus. The predicted amino acid sequence of the protein shows regions of strong identity with those of three other rat acyltransferases, namely, liver CPT II, liver carnitine octanoyltransferase, and brain choline acetyltransferase. The findings provide the first insight into the structure of a CPT I isoform. They also establish unequivocally that CPT I and CPT II are distinct proteins and that inhibitors of CPT I interact within its catalytic domain, not with an associated regulatory component.
我们报道了编码大鼠肝脏肉碱棕榈酰转移酶I（CPT I）的全长cDNA的分离与鉴定。对应于源自大鼠肝脏线粒体丙二酰辅酶A/依托莫昔-CoA结合蛋白的两个胰蛋白酶肽段的寡核苷酸（埃塞尔，V.，桑岛，M.，布里顿，C. H.，克里希南，K.，福斯特，D. W.，和麦加里，J. D.（1993年）《生物化学杂志》268卷，5810 - 5816页）被用于筛选构建在质粒克隆载体pcDV中的大鼠肝脏cDNA文库。获得的克隆包含一个102个核苷酸的5'非翻译区、一个2319个碱基的单一开放阅读框，预测编码一个773个氨基酸的蛋白质（分子量 = 88,150），以及一个1957个核苷酸的3'非翻译片段，其后是聚腺苷酸尾巴。该cDNA的一个0.9千碱基片段在大鼠肝脏中识别出一种单一的mRNA物种（大小约为4.7千碱基）。cDNA的身份通过以下发现得以证实：（i）开放阅读框编码了原始蛋白质中发现的所有四个肽段；（ii）将亚克隆到表达载体pCMV6中的cDNA转染COS细胞，导致丙二酰辅酶A和依托莫昔-CoA敏感的CPT活性选择性地诱导10 - 20倍；（iii）通过针对起始材料产生的抗体在蛋白质印迹上很容易检测到过表达的产物。新合成的酶似乎通过一个前导肽靶向线粒体外膜，并且成熟蛋白通过其氨基末端附近存在的一段20个氨基酸实现膜锚定。该蛋白质的预测氨基酸序列显示与其他三种大鼠酰基转移酶，即肝脏CPT II、肝脏肉碱辛酰转移酶和脑胆碱乙酰转移酶的氨基酸序列有高度同源区域。这些发现首次揭示了CPT I同工型的结构。它们还明确证实CPT I和CPT II是不同的蛋白质，并且CPT I抑制剂在其催化结构域内相互作用，而不是与相关的调节成分相互作用。
