Darnis S, Juge N, Guo X J, Marchis-Mouren G, Puigserver A, Chaix J C
Laboratoire de Biochimie et Biologie de la Nutrition, CNRS-UPRESA 6033, Faculté des Sciences et Techniques de St Jérôme, LBBN case 342, Université d'Aix-Marseille, Avenue Escadrille Normandie-Niemen, F-13397, Marseille cedex 20, France.
Biochim Biophys Acta. 1999 Mar 19;1430(2):281-9. doi: 10.1016/s0167-4838(99)00011-4.
A cDNA library was constructed in a Uni-ZAP XR vector using mRNA isolated from porcine pancreas. A full-length alpha-amylase cDNA was obtained using a combination of library screening and nested polymerase chain reaction. Sequencing of the clone revealed a 1536-nucleotide (nt) open reading frame encoding a protein of 496 amino acid (aa) residues with a signal peptide of 15 aa. The calculated molecular mass of the enzyme was 55354 Da, in accordance with those of the purified porcine pancreatic alpha-amylase forms (PPAI and PPAII) as determined by mass spectrometry. A comparison of the deduced aa sequence with published peptidic sequences of PPAI identified a number of mismatches. The sequence of the cDNA reported here provides a sequence reference for PPA in excellent agreement with the refined three-dimensional structures of both PPAI and PPAII. No evidence for a second variant was found in the cDNA library and it is most likely that PPAI and PPAII are two forms of the same protein. The primary structure of PPA shows high homology with human, mouse and rat pancreatic alpha-amylases. The 304-310 region, corresponding to a mobile loop involved in substrate binding and processing near the active site, is fully conserved.
使用从猪胰腺分离的mRNA构建了Uni-ZAP XR载体中的cDNA文库。通过文库筛选和巢式聚合酶链反应相结合的方法获得了全长α-淀粉酶cDNA。对该克隆进行测序，发现一个1536个核苷酸（nt）的开放阅读框，编码一个由496个氨基酸（aa）残基组成的蛋白质，带有一个15个aa的信号肽。根据质谱测定的纯化猪胰腺α-淀粉酶形式（PPAI和PPAII）的分子量，计算出该酶的分子量为55354 Da。将推导的aa序列与已发表的PPAI肽序列进行比较，发现了一些错配。本文报道的cDNA序列为PPA提供了一个序列参考，与PPAI和PPAII的精细三维结构高度一致。在cDNA文库中未发现第二种变体的证据，很可能PPAI和PPAII是同一蛋白质的两种形式。PPA的一级结构与人类、小鼠和大鼠的胰腺α-淀粉酶具有高度同源性。对应于活性位点附近参与底物结合和加工的可移动环的304-310区域完全保守。
