Koyama K, Yokoyama M, Koike T, Ohtsuki K, Ishida N
J Biochem. 1984 Apr;95(4):925-35. doi: 10.1093/oxfordjournals.jbchem.a134720.
A phosphate-incorporating protein has been highly purified from the cytosol of Ehrlich ascites tumor cells (EAT cells). The nitrocellulose membrane method was used to follow the progress of the purification by quantitation of the [32P]phosphorylated form of the protein. The purified protein was identified as an NDP-kinase since it exhibited NDP-kinase activity and had enzyme characteristics in common with other NDP-kinases from various mammalian cells. The purified NDP-kinase was found to have a molecular weight of approximately 76,000 daltons. Moreover, the enzyme appears to consist of two distinct polypeptides (18,000 and 20,000 daltons). This enzyme contained 19 amino acids, with high levels of glycine (9.8%) and lysine (9.0%). The enzyme rapidly formed a [32P]phosphoenzyme when incubated with [gamma-32P]ATP in the presence of Mg2+ (1 mM) at the optimum pH of 7.5 even at low temperature (below 4 degrees C). This phosphoenzyme is an enzyme-bound, high-energy-phosphate intermediate, because ATP was formed from it on incubation with ADP in the presence of Mg2+ (1 mM). This finding suggests that the phosphoenzyme functions as an intermediate in NDP-kinase action.
一种含磷酸的蛋白质已从艾氏腹水瘤细胞（EAT细胞）的胞质溶胶中高度纯化出来。采用硝酸纤维素膜法，通过对该蛋白质的[32P]磷酸化形式进行定量来跟踪纯化进程。纯化后的蛋白质被鉴定为核苷二磷酸激酶，因为它具有核苷二磷酸激酶活性，且具有与来自各种哺乳动物细胞的其他核苷二磷酸激酶相同的酶学特性。发现纯化后的核苷二磷酸激酶分子量约为76,000道尔顿。此外，该酶似乎由两种不同的多肽（18,000和20,000道尔顿）组成。这种酶含有19种氨基酸，其中甘氨酸（9.8%）和赖氨酸（9.0%）含量较高。在1 mM Mg2+存在下，于7.5的最适pH值下，即使在低温（4℃以下）条件下，该酶与[γ-32P]ATP孵育时也能迅速形成[32P]磷酸酶。这种磷酸酶是一种与酶结合的高能磷酸中间体，因为在1 mM Mg2+存在下与ADP孵育时，它能生成ATP。这一发现表明，磷酸酶在核苷二磷酸激酶的作用中起中间体的作用。
