Weldon S L, Rando A, Matathias A S, Hod Y, Kalonick P A, Savon S, Cook J S, Hanson R W
Department of Biochemistry, School of Medicine, Case Western Reserve University, Cleveland, Ohio 44106.
J Biol Chem. 1990 May 5;265(13):7308-17.
The amino acid sequence of the mitochondrial form of phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) (PEPCK-M) from the chicken was deduced from the 3571 nucleotide sequence of three overlapping cDNA clones. The derived protein sequence, which includes 607 amino acids of the mature enzyme and a leader sequence, was aligned with nine tryptic peptides of PEPCK-M and the primary sequence of the cytosolic form of PEPCK from chicken. Secondary structure predictions for the two PEPCK isozymes indicated similar packing elements of conserved, hydrophobic beta strands in the central core of the primary sequence. This core protein, which contained three GTP-binding consensus elements, was 80% identical in the two chicken isozymes, although the overall level of identity was only 63% for amino acids and 60% for nucleotides. The untranslated regions of the two cDNAs were dissimilar, although both mRNAs have potential for significant secondary structure. The PEPCK-M mRNA contained several G-C-rich regions which demonstrated free energies of formation in dyad symmetry programs up to -70 kcal/mol. The 1.6-kilobase (kb) 3'-untranslated region contained several repeat elements including one of 11 base pairs, which was present 30 times; but, a signal sequence for polyadenylation was not present. Each of the three PEPCK-M cDNA clones recognized two mRNAs of 4.2 and 3.4 kb in the livers and kidneys of starved or normally fed chickens. However, the level of these two related PEPCK-M mRNAs changed in response to cAMP treatment, with the larger mRNA predominant at 20 and 160 min and the 3.4-kb mRNA present at intermediate times. In contrast, the level of the 2.8-kb PEPCK-C mRNA increased dramatically upon addition of the cyclic nucleotide, particularly in the liver where it was not detected without cAMP induction. Thus, PEPCK-M and PEPCK-C, clearly represented the products of two distinct genes, which were distinguished by altered protein sequences and non-cross-hybridizing, differentially regulated mRNAs.
通过三个重叠cDNA克隆的3571个核苷酸序列推导得出鸡的线粒体磷酸烯醇丙酮酸羧激酶（GTP）（EC 4.1.1.32）（PEPCK-M）的氨基酸序列。推导得到的蛋白质序列包括成熟酶的607个氨基酸和一个前导序列，将其与PEPCK-M的九个胰蛋白酶肽段以及鸡的胞质型PEPCK的一级序列进行比对。对两种PEPCK同工酶的二级结构预测表明，在一级序列的中央核心区域，保守的疏水β链具有相似的堆积元件。该核心蛋白包含三个GTP结合共有元件，在两种鸡同工酶中80%相同，尽管氨基酸的总体同一性水平仅为63%，核苷酸的同一性水平为60%。两种cDNA的非翻译区不同，尽管两种mRNA都具有形成显著二级结构的潜力。PEPCK-M mRNA包含几个富含G-C的区域，在二元对称程序中显示出高达-70千卡/摩尔的形成自由能。1.6千碱基（kb）的3'非翻译区包含几个重复元件，包括一个11个碱基对的元件，该元件出现了30次；但是，不存在聚腺苷酸化信号序列。三个PEPCK-M cDNA克隆中的每一个都能识别饥饿或正常喂养的鸡的肝脏和肾脏中4.2 kb和3.4 kb的两种mRNA。然而，这两种相关的PEPCK-M mRNA的水平会因cAMP处理而发生变化，较大的mRNA在20分钟和160分钟时占主导，3.4 kb的mRNA在中间时间出现。相比之下，加入环核苷酸后，2.8 kb的PEPCK-C mRNA水平显著增加，特别是在肝脏中，没有cAMP诱导时未检测到该mRNA。因此，PEPCK-M和PEPCK-C显然代表两个不同基因的产物，它们通过改变的蛋白质序列和非交叉杂交、差异调节的mRNA来区分。
