Woeltje K F, Esser V, Weis B C, Sen A, Cox W F, McPhaul M J, Slaughter C A, Foster D W, McGarry J D
Department of Internal Medicine, University of Texas Southwestern Medical Center, Southwestern Medical School, Dallas 75235.
J Biol Chem. 1990 Jun 25;265(18):10720-5.
We report the isolation and characterization of a full-length cDNA encoding rat liver carnitine palmitoyltransferase II (CPT II). Beginning with the purified protein CNBr fragments were generated and sequenced. Corresponding oligonucleotides were used to screen a rat liver cDNA library constructed in the plasmid cloning vector, pcDV. The clone ultimately obtained consisted of a 62 nucleotide 5'-untranslated region, a single open reading frame of 1,974 bases predicting a protein of 658 amino acids (Mr = 74,119), and a 3'-untranslated segment of 260 nucleotides followed by the poly (A) tail. The identity of the cDNA was confirmed by the findings that (a) the open reading frame encoded all three peptides found in the original protein; (b) a fourth peptide synthesized from a portion of the deduced amino acid sequence and used to immunize a rabbit resulted in the generation of an antibody that recognized pure CPT II on a Western blot; (c) in vitro transcription and translation of the cDNA (ligated into pBlue-script KS (+] generated a protein that was specifically immunoprecipitated by anti-CPT II antibody and having a Mr slightly greater than that of mature CPT II; (d) transfection of COS cells with the cDNA subcloned into the expression vector, pCMV4, resulted in a 6-fold induction of mitochondrial CPT II catalytic activity. It seems likely that the de novo synthesized enzyme gains entry into the mitochondrion via a targeting peptide that is subsequently cleaved. The mature protein probably associates (relatively loosely) with the inner membrane through a limited number of membrane spanning domains. The predicted amino acid sequence of CPT II shows strong identity with those of two other acyltransferases, namely, rat liver peroxisomal carnitine octanoyltransferase and porcine choline acetyltransferase.
我们报道了编码大鼠肝脏肉碱棕榈酰转移酶II（CPT II）的全长cDNA的分离和特性分析。从纯化的蛋白质开始，生成并测序了溴化氰片段。相应的寡核苷酸用于筛选构建在质粒克隆载体pcDV中的大鼠肝脏cDNA文库。最终获得的克隆由一个62个核苷酸的5'-非翻译区、一个1974个碱基的单一开放阅读框（预测编码一个658个氨基酸的蛋白质，Mr = 74,119）、一个260个核苷酸的3'-非翻译区以及后面的聚腺苷酸尾巴组成。cDNA的身份通过以下发现得到证实：（a）开放阅读框编码了原始蛋白质中发现的所有三种肽段；（b）从推导的氨基酸序列的一部分合成的第四种肽段用于免疫兔子，产生了一种抗体，该抗体在蛋白质印迹上能识别纯CPT II；（c）cDNA（连接到pBlue-script KS(+)中）的体外转录和翻译产生了一种蛋白质，该蛋白质能被抗CPT II抗体特异性免疫沉淀，其Mr略大于成熟CPT II；（d）将亚克隆到表达载体pCMV4中的cDNA转染COS细胞，导致线粒体CPT II催化活性诱导增加6倍。新合成的酶似乎通过随后被切割的靶向肽进入线粒体。成熟蛋白质可能通过有限数量的跨膜结构域（相对松散地）与内膜结合。CPT II的预测氨基酸序列与另外两种酰基转移酶，即大鼠肝脏过氧化物酶体肉碱辛酰转移酶和猪胆碱乙酰转移酶的氨基酸序列具有很强的同源性。
