Purification of a novel enzyme, 5'-nucleotide phosphodiesterase, and studies on its physiological role and relation to tumor.

我们发现许多肿瘤细胞系表达一种5′-核苷酸磷酸二酯酶（5′- npase），其性质与正常组织的酶明显不同。该酶具有5′- npase活性，经多步骤柱层析纯化得到，sds -聚丙烯酰胺凝胶电泳显示单带。与正常细胞型酶不同，肿瘤细胞型酶是一种可溶性蛋白，最适pH为7.5，SDS-PAGE估计其分子量为6.7 kDa。该酶被二硫苏糖醇激活，被n -乙基马来酰亚胺抑制，而正常细胞型酶被二硫苏糖醇抑制。该酶可裂解胸苷5′-单磷酸对硝基苯基酯（TMP-NP），但不能水解磷酸二酯酶的其他显色底物，也不能水解正常组织中被5′裂解的各种核苷酸的焦磷酸键。但是，它能水解二核苷酸生成5'-磷酸，并且在2',5'-磷酸二酯键上比在3',5'-磷酸二酯键上更活跃。这些结果表明，埃利希腹水癌细胞的TMP-NP分裂酶是一种2',5'-磷酸二酯酶。我们正试图从埃利希腹水肿瘤细胞中获得一种针对这种酶的单克隆抗体，以了解这种酶是否可以用于癌症的诊断。然而，到目前为止，我们还没有成功。

We have found that many tumor cell lines express a 5'-nucleotide phosphodiesterase (5'-NPDase) with properties clearly distinguishable from enzymes of normal tissues. Such an enzyme with 5'-NPDase activity was purified from Ehrlich ascites carcinoma after several steps of column chromatography to show a single band on SDS-polyacrylamide gel electrophoresis. Unlike the normal cell type enzyme, the tumor cell type enzyme is a soluble protein, has a pH optimum of 7.5, and the molecular mass estimated by SDS-PAGE is 6.7 kDa. The enzyme is activated by dithiothreitol and inhibited by N-ethylmaleimide, whereas the normal cell type enzyme was inhibited by dithiothreitol. The enzyme cleaves thymidine 5'-mono- phosphate p-nitrophenyl ester (TMP-NP) but does not hydrolyze other chromogenic substrate for phosphodiesterases, nor pyrophosphate bond of various nucleotides which are cleaved by 5' of normal tissues. But, it hydrolyzes dinucleotides to from 5'-phosphates, and is more active on 2', 5'-than on 3', 5'-phosphodiester bonds. These results indicate that the TMP-NP splitting enzyme in Ehrlich ascites carcinoma cell is a 2', 5'-phosphodiesterase. We are trying to get a monoclonal antibody against this enzyme from Ehrlich ascites tumor cells to know whether this enzyme could be used for diagnosis of cancer. However, we have not succeeded in it so far.