Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II.
Cloning, sequencing, and expression of a cDNA encoding rat liver mitochondrial carnitine palmitoyltransferase II.
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DOI：
10.1016/s0021-9258(18)87006-5
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发表时间：
1990-06
期刊：
影响因子：
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通讯作者：
K. Woeltje;V. Esser;B. Weis;A. Sen;W. F. Cox;M. McPhaul;C. Slaughter;D. W. Foster;J. McGarry
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文献类型：
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作者：
K. Woeltje;V. Esser;B. Weis;A. Sen;W. F. Cox;M. McPhaul;C. Slaughter;D. W. Foster;J. McGarry
我们报告的分离和鉴定的全长cDNA编码大鼠肝脏肉毒碱棕榈酰转移酶II（CPT II）。从纯化的蛋白质开始，生成CNBr片段并进行测序。使用相应的寡核苷酸筛选在质粒克隆载体pcDV中构建的大鼠肝脏cDNA文库。最终获得的克隆由62个核苷酸的5 '-非翻译区、预测658个氨基酸的蛋白质的1，974个碱基的单个开放阅读框（Mr = 74，119）和260个核苷酸的3'-非翻译片段以及随后的聚腺苷酸尾组成。通过以下发现证实了cDNA的同一性：（a）开放阅读框编码在原始蛋白质中发现的所有三种肽;（B）从推导的氨基酸序列的一部分合成的第四种肽，并用于免疫兔，导致产生在Western印迹上识别纯CPT II的抗体;（c）cDNA的体外转录和翻译（连接到pBlue-script KS（+）中产生了一种蛋白质，该蛋白质被抗CPT II抗体特异性免疫沉淀，并且其Mr略大于成熟CPT II的Mr;（d）用亚克隆到表达载体pCMV 4中的cDNA转染COS细胞，导致线粒体CPT II催化活性的6倍诱导。似乎可能的是，从头合成的酶通过随后被切割的靶向肽进入到靶向子中。成熟蛋白可能通过有限数量的跨膜结构域与内膜结合（相对松散）。预测的CPT II的氨基酸序列与其他两个酰基转移酶，即大鼠肝脏过氧化物酶体肉毒碱辛酰基转移酶和猪胆碱乙酰转移酶的序列具有很强的一致性。
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.