Lambeth D O, Mehus J G, Ivey M A, Milavetz B I
Department of Biochemistry and Molecular Biology, University of North Dakota School of Medicine and Health Sciences, Grand Forks, North Dakota 58202, USA.
J Biol Chem. 1997 Sep 26;272(39):24604-11. doi: 10.1074/jbc.272.39.24604.
Nucleoside-diphosphate kinase (NDP kinase) from the matrix space of mitochondria in pigeon liver was purified to homogeneity. Degenerate oligonucleotide primers to the N-terminal sequence of the purified protein and the region containing the active site histidine were used in reverse transcriptase-polymerase chain reaction to obtain a major portion of the coding sequence for the mature protein. The sequences of the C and N termini of the mature protein, and eight residues in the signal peptide, were obtained by rapid amplification of cDNA end procedures. The entire coding sequence of a cytosolic form of NDP kinase was also determined. Both isoforms, which share 53% sequence identity, possess the characteristically conserved regions of known NDP kinases. The mature mitochondrial NDP kinase protein migrates in molecular sieving columns with an apparent molecular mass of about 66 kDa. It shows very high thermal stability even though it lacks the proline residue in the killer of prune loop, and the Tyr/Glu C termini that are important in stabilizing other NDP kinases. The affinity of the mitochondrial isoform for adenine and guanine nucleotides is much higher than for pyrimidine nucleotides, but the enzyme is especially susceptible to substrate inhibition by GDP. Semi-quantitative reverse transcriptase-polymerase chain reaction showed that the relative levels of expression of the mitochondrial isoform are liver > kidney >> heart = brain > breast muscle. The cytosolic isoform is strongly and approximately equally expressed in these same five tissues. This work is the first characterization of a NDP kinase isoform that is found in the matrix space of mitochondria.
对鸽肝线粒体基质空间中的核苷二磷酸激酶（NDP激酶）进行了纯化，使其达到同质。利用针对纯化蛋白N端序列和包含活性位点组氨酸区域的简并寡核苷酸引物，通过逆转录聚合酶链反应获得了成熟蛋白编码序列的主要部分。通过cDNA末端快速扩增程序获得了成熟蛋白C端和N端的序列以及信号肽中的八个残基。还确定了胞质形式NDP激酶的完整编码序列。两种同工型的序列一致性为53%，均具有已知NDP激酶的特征性保守区域。成熟的线粒体NDP激酶蛋白在分子筛柱中的迁移表观分子量约为66 kDa。尽管它在“梅干杀手”环中缺乏脯氨酸残基以及在稳定其他NDP激酶中起重要作用的Tyr/Glu C端，但它仍表现出非常高的热稳定性。线粒体同工型对腺嘌呤和鸟嘌呤核苷酸的亲和力远高于对嘧啶核苷酸的亲和力，但该酶特别容易受到GDP的底物抑制。半定量逆转录聚合酶链反应表明，线粒体同工型的相对表达水平为肝脏＞肾脏＞＞心脏 = 大脑＞胸肌。胞质同工型在这相同的五个组织中强烈且大致等量表达。这项工作首次对在线粒体基质空间中发现 的NDP激酶同工型进行了表征。
