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

新型酶5-核苷酸磷酸二酯酶的纯化及其生理作用和与肿瘤的关系研究。

• 批准号: 63571034
• 负责人: SUZUKI Hideo
• 金额: $ 1.47万
• 依托单位: The University of Tokyo
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1990
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63571034/
• 关键词: Tumor cell specific; 5'-Nucleotide phosphodiesterase; Ehrlich ascites tumor cells; Purification of a enzyme; Monoclonal antibody; 5'ーヌクレオチドホスホジエステラ-ゼ; 5′-ヌクレオチドホスホジエステラ-ゼ; 基質特異性; 5'-ヌクレオチドホスホジエステラーゼ; アルカリホスファターゼ; 癌細胞

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.

我们发现许多肿瘤细胞株表达5‘-核苷酸磷酸二酯酶(5’-NPDase)，其性质与正常组织的酶明显不同。这种具有5‘-NPDase活性的酶是从艾氏腹水癌中提纯的，经过几步柱层析，在SDS-聚丙烯酰胺凝胶电泳法上显示一条单一的条带。与正常细胞型酶不同，肿瘤细胞型酶是一种可溶性蛋白质，最适pH为7.5，经SDS-PAGE测得其相对分子质量为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。该酶可裂解胸腺嘧啶核苷5‘-单磷酸对硝基苯酯(TMP-NP)，但不能水解其他磷酸二酯酶的显色底物，也不能分解被正常组织5’端裂解的各种核苷酸的焦磷酸键。但是，它将二核苷酸水解成5‘-磷酸，并且在2’，5‘-键上比在3’，5‘-磷酸二酯键上更有活性。这些结果表明，艾氏腹水癌细胞的TMP-NP裂解酶是一种2‘，5’-磷酸二酯酶。我们正试图从Ehrlich腹水肿瘤细胞中获得针对该酶的单抗，以了解该酶是否可用于癌症诊断。然而，到目前为止，我们还没有成功。

项目成果

FUKAZAWA, H.: "5'-Nucleotide phosphodiesterase and alkaline phosphatase in tumor cells ; Evidence for existence of novel species in the cytosol." Biochim. Biophys. Acta. 966. 99-106 (1988)

FUKAZAWA, H.：“肿瘤细胞中的 5-核苷酸磷酸二酯酶和碱性磷酸酶；细胞质中存在新物种的证据。”

HIDESUKE FUKAZAWA: "A nucleotide phosphodiesterase with preference for 2',5'-phosphodiester bonds from Ehrlich ascites carcinoma." Biochim.Biophys.Acta. 991. 272-275 (1989)

HIDESUKE FUKAZAWA：“一种优先选择来自艾利希腹水癌的 2,5-磷酸二酯键的核苷酸磷酸二酯酶。”

FUKAZAWA HIDESUKE: "5'ーNucleotide phosphodiesterase and alkaline phesphatase in tumor cells;Evidence for existence of novel species in the cytosol." Biochim.Biophys.Acta. 966. 99-106 (1988)

Fukazawa HIDESUKE：“肿瘤细胞中的 5-核苷酸磷酸二酯酶和碱性磷酸酶；细胞质中存在新物种的证据。” 966. 99-106 (1988)

HIDESUKE,FUKAZAWA: Biochim.Biophys.Acta. 966. 99-106 (1988)

深泽英介：生物化学、生物物理学学报。

FUKAZAWA HIDESUKE: "A nucleotide phosphodiesterase with proference for 2',5'ーphosphodiesterase bonds from Ehrlich ascites carcinoma." Biochim.Bioohys.Acta. 991. 272-275 (1989)

FUKAZAWA HIDESUKE：“一种来自艾利希腹水癌的 2,5-磷酸二酯酶键的核苷酸磷酸二酯酶。”Biochim.Biohys.Acta。