Hu C W, Lau K S, Griffin T A, Chuang J L, Fisher C W, Cox R P, Chuang D T
Department of Medicine, Veterans Administration Medical Center, Cleveland, Ohio 44106.
J Biol Chem. 1988 Jun 25;263(18):9007-14.
A cDNA clone encoding the entire decarboxylase (E1)alpha precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex has been isolated from a lambda ZAP library prepared from bovine liver poly(A)+ RNA. Nucleotide sequencing indicates that this E1 alpha cDNA clone is 1821 base pairs (bp) in length with an open reading frame of 1365 bp and a 3'-untranslated region of 356 bp. A polyadenylation signal of the type AATAAA is located 27 bp upstream of the start of a poly(A)+ tail. There is a pair of identical 32-bp direct repeats of unknown function at the 5'-end of the cDNA. The bovine E1 alpha cDNA encodes a leader peptide of 55 residues including three candidate initiation methionines, and a mature E1 alpha of 400 amino acids with a calculated Mr of 45,385. The deduced primary structure shows the published peptide sequences flanking the two phosphorylation sites and the amino-terminal sequence (residues 1-32) of bovine E1 alpha determined in this study. The phosphoserine-bearing regions appear to be homologous between bovine E1 alpha and human pyruvate decarboxylase-alpha subunits, with respect to both amino acid identity and the position in each polypeptide chain. Northern blot analysis using the bovine E1 alpha cDNA as probe shows the presence of a single species of E1 alpha mRNA (2 kilobase pairs) in bovine liver, human placenta, and skin fibroblasts. Moreover, the E1 alpha mRNA exists in normal size and quantity in cultured fibroblasts derived from a maple-syrup-urine-disease homozygote deficient in E1 activity. The results preclude a defect in the transcription and processing of E1 alpha mRNA in these maple-syrup-urine-disease cells. Studies with 3T3-L1 cells show that a single species of E1 alpha mRNA (2 kilobase pairs) is expressed in the cells and that contents of the murine E1 alpha mRNA and subunit are markedly elevated during the differentiation of 3T3-L1 preadipocytes into adipocytes. The results indicate that the induction of murine E1 activity during adipocyte differentiation occurs at the pretranslational level.
从以牛肝聚腺苷酸加尾RNA构建的λZAP文库中分离出了一个编码牛支链α-酮酸脱氢酶复合体全脱羧酶（E1）α前体的cDNA克隆。核苷酸测序表明，这个E1α cDNA克隆长度为1821个碱基对（bp），有一个1365 bp的开放阅读框和一个356 bp的3'-非翻译区。AATAAA类型的聚腺苷酸化信号位于聚腺苷酸加尾起始点上游27 bp处。在cDNA的5'-末端有一对功能未知的32 bp相同直接重复序列。牛E1α cDNA编码一个含55个残基的前导肽，包括三个候选起始甲硫氨酸，以及一个400个氨基酸的成熟E1α，计算的分子量为45385。推导的一级结构显示了本研究中确定的牛E1α两个磷酸化位点两侧的已发表肽序列和氨基末端序列（第1至32位残基）。就氨基酸同一性和每条多肽链中的位置而言，含磷酸丝氨酸区域在牛E1α和人丙酮酸脱羧酶α亚基之间似乎是同源的。以牛E1α cDNA为探针进行的Northern印迹分析表明，在牛肝、人胎盘和皮肤成纤维细胞中存在单一物种的E1α mRNA（2千碱基对）。此外，在源自缺乏E1活性的枫糖尿症纯合子的培养成纤维细胞中，E1α mRNA的大小和数量正常。这些结果排除了这些枫糖尿症细胞中E1α mRNA转录和加工存在缺陷的可能性。对3T3-L1细胞的研究表明，细胞中表达单一物种的E1α mRNA（2千碱基对），并且在3T3-L1前脂肪细胞分化为脂肪细胞的过程中，小鼠E1α mRNA和亚基的含量显著升高。结果表明，脂肪细胞分化过程中小鼠E1活性的诱导发生在翻译前水平。
