Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: Molecular identification, kinetic characterization and biochemical properties
Nucleotide metabolizing ecto-enzymes in Walker 256 tumor cells: Molecular identification, kinetic characterization and biochemical properties
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DOI：
10.1016/j.lfs.2006.11.024
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发表时间：
2007-02-13
期刊：
影响因子：
6.1
通讯作者：
Sarkis, Joao J. F.
中科院分区：
文献类型：
--
作者：
Buffon, Andreia;Ribeiro, Vanessa B.;Sarkis, Joao J. F.
在本研究中，我们描述了Walker 256细胞中e - ntpase和5'-核苷酸酶的分子鉴定、动力学表征和生化特性。ATP、ADP和AMP水解的最适pH为6.5 ~ 8.0，绝对需要二价阳离子（Mg2+ > Ca2+）。在高浓度叠氮化钠和0.5 mM氯化钆的存在下，ATP和ADP的水解被显著抑制。这些活性对atp酶、腺苷酸激酶和碱性磷酸酶经典抑制剂不敏感。k值分别为464.2 + / - 86.6μM(意思是+ / - SEM, n = 4), 31 + / - 137.0μM(意思是+ / - SEM, n = 5)和44.8 + / - 10.2μM(意思是+ / - SEM, n = 4),和vmax值分别为655.0 + / - 94.6 (+ / - SEM, n = 4), 236.3 + / - 27.2 (+ / - SEM, n = 5)和177.6 + / - 13.8(意思是SEM, n = 5) nmol无机磷酸盐的最小值(1)毫克的蛋白质(1)ATP、ADP和AMP,分别。通过RT-PCR分析，我们鉴定了两个外核苷三磷酸二磷酸水解酶家族成员（ntpase 2和5）和一个5'-核苷酸酶的mRNA。Walker 256肿瘤细胞中ntpases和5-核苷酸酶的存在可能在细胞外调节腺嘌呤核苷酸/腺嘌呤核苷的比例，从而促进肿瘤生长方面发挥重要作用。(c) 2006爱思唯尔公司版权所有。
In this study we describe the molecular identification, kinetic characterization and biochemical properties of an E-NTPDase and an 5'-nucleotidase in Walker 256 cells. For the ATP, ADP and AMP hydrolysis there were optimum pH in the range 6.5-8.0, and absolute requirement for divalent cations (Mg2+ > Ca2+). A significant inhibition of ATP and ADP hydrolysis was observed in the presence of high concentrations of sodium azide and 0.5 mM of Gadolinium chloride. These activities were insensitive to ATPase, adenylate kinase and alkaline phosphatase classical inhibitors. The K. values were 464.2 +/- 86.6 mu M (mean +/- SEM, n = 4), 137.0 +/- 31 mu M (mean +/- SEM, n = 5) and 44.8 +/- 10.2 mu M (mean +/- SEM, n = 4), and V-max values were 655.0 +/- 94.6 (mean +/- SEM, n = 4), 236.3 +/- 27.2 (mean +/- SEM, n = 5) and 177.6 +/- 13.8 (mean SEM, n = 5) nmol of inorganic phosphate min(-1) mg of protein(-1) for ATP, ADP and AMP, respectively. Using RT-PCR analysis we identified the mRNA of two members of the ecto-nucleoside triphosphate diphosphohydrolase family (NTPDase 2 and 5) and a 5'-nucleotidase. The presence of NTPDases and 5-nucleotidase enzymes in Walker 256 tumor cells may be important to regulate the ratio adenine nucleotides/adenine nucleoside extracellularly, therefore motivating tumor growth. (c) 2006 Elsevier Inc. All rights reserved.