Jitrapakdee S, Booker G W, Cassady A I, Wallace J C
Department of Biochemistry, University of Adelaide, South Australia, Australia.
Biochem J. 1996 Jun 1;316 ( Pt 2)(Pt 2):631-7. doi: 10.1042/bj3160631.
Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated M(r) of 129848. This is flanked by a 5' untranslated region of 66 bp and a 3' untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249-10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.
通过筛选肝脏cDNA文库以及对肝脏总RNA进行cDNA末端快速扩增聚合酶链反应，已分离出编码大鼠肝脏丙酮酸羧化酶（PC）的重叠克隆。大鼠PC cDNA序列包含一个3537个核苷酸的开放阅读框，编码一个1178个氨基酸的多肽，计算所得的分子量为129848。其两侧分别是一个66 bp的5'非翻译区和一个421 bp的3'非翻译区，后者包括poly(A)尾。推断出的蛋白质序列与小鼠PC的同源性为96.6%，与人类PC的同源性为96.3%，与蚊子PC的同源性为68.4%，与酵母PC同工酶PC1和PC2的同源性为53.5%。基于部分蛋白水解以及与其他生物体（酵母、蚊子、小鼠和人类）的PC以及其他生物素酶的序列同源性，已鉴定出三个功能结构域，即生物素羧化结构域、转羧化结构域和生物素结构域。与大肠杆菌乙酰辅酶A羧化酶生物素羧化酶亚基的已知结构[Waldrop, Rayment和Holden（1994年）《生物化学》33, 10249 - 10256]进行比较，突出了11个高度保守残基的功能重要性。Northern分析显示，PC mRNA在大鼠肝脏、肾脏、脂肪组织和大脑中高表达，在心脏、肾上腺和泌乳乳腺中中等表达，在脾脏和骨骼肌中低表达。
