Haselbeck R J, Colman R F, McAlister-Henn L
Department of Biological Chemistry, School of Medicine, University of California, Irvine 92717.
Biochemistry. 1992 Jul 14;31(27):6219-23. doi: 10.1021/bi00142a007.
The cDNA for porcine mitochondrial NADP-specific isocitrate dehydrogenase was isolated from a lambda gt11 library using polymerase chain reaction. Translation of the DNA sequence gave a 413-residue amino acid sequence and a calculated molecular weight of 46,600 for the mature polypeptide. Previously determined peptide sequences for the amino terminus and for internal tryptic peptides were all contained within the translated sequence. The porcine protein was found to share 63% residue identity with yeast mitochondrial NADP-specific isocitrate dehydrogenase and to be immunoreactive with an antiserum against the yeast protein. Highly conserved regions include residues which have been implicated in substrate and cofactor binding in previous studies of the porcine enzyme. The two eucaryotic enzymes exhibit only minimal homology with the NADP-dependent isocitrate dehydrogenase from Escherichia coli, with the exception of a striking conservation of residues implicated in formation of the metal-isocitrate site of the procaryotic enzyme.
利用聚合酶链反应从λgt11文库中分离出猪线粒体NADP特异性异柠檬酸脱氢酶的cDNA。DNA序列的翻译产生了一个413个残基的氨基酸序列，成熟多肽的计算分子量为46,600。先前确定的氨基末端和内部胰蛋白酶肽的肽序列都包含在翻译序列中。发现猪蛋白与酵母线粒体NADP特异性异柠檬酸脱氢酶具有63%的残基同一性，并且与抗酵母蛋白的抗血清具有免疫反应性。高度保守的区域包括在先前对猪酶的研究中涉及底物和辅因子结合的残基。这两种真核酶与大肠杆菌的NADP依赖性异柠檬酸脱氢酶仅表现出最小的同源性，除了在原核酶的金属异柠檬酸位点形成中涉及的残基有显著的保守性。
