Ceramide glycanase activities in human cancer cells.
Ceramide glycanase activities in human cancer cells.
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人类癌细胞中的神经酰胺聚糖酶活性。
DOI：
10.1023/a:1020220524180
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发表时间：
1999
影响因子：
4
通讯作者：
Basu,S
中科院分区：
文献类型：
--
作者：
Basu,M;Kelly,P;O'Donnell,P;Miguel,M;Bradley,M;Sonnino,S;Banerjee,S;Basu,S
在不同的人类肿瘤细胞（结肠癌 Colo-205；神经母细胞瘤 IMR-32；乳腺癌细胞系 SKBr3 和 MCF7）中检测到了神经酰胺聚糖酶 (CGase) 活性。然而，与之前报道的其他哺乳动物组织中存在的酶活性水平相比，这些细胞中的酶活性水平较低（Basu, M.、Kelly, P.、Girzadas, M. A.、Li, Z. 和 Basu, S.Methods Enzymol.（出版中））。大部分 CGase 活性存在于从所有这些细胞系和组织中分离的 100,000g 可溶性上清液级分中。使用可溶性酶，发现最佳 CGase 活性的要求与先前对大鼠和兔组织的观察结果一致（Basu, M.、Dastgheib, S.、Girzadas, M. A.、O'Donnell, P. H.、Westervelt, C. W.、Li, Z.、Inokuchi, J. I. 和 Basu, S. (1998)Acta Pol.生物化学。42：327）。 Colo-205 和 IMR-32 细胞的 CGase 活性在蛋白质与去垢剂比率为 1 时达到最佳。所有哺乳动物 CGase，包括人类癌细胞，在乙酸钠缓冲液中显示出 5.5 至 5.8 之间的最佳 pH 值。研究发现癌细胞的 CGase 活性与阳离子无关；然而，汞、锌和铜离子似乎显着抑制两种肿瘤细胞系中的酶活性。所有不同来源的 CGase 活性的汞离子抑制表明 CGase 蛋白中可能存在结构同源性。本研究中使用了放射性标记底物，标记在鞘氨醇双键或在鞘氨醇 3 位上而不修饰鞘氨醇双键。两者都是活性底物，所有酶制剂均从不同癌细胞中分离出来（表观 Km，对于 nLcOse5[3H-DT]Cer 为 500 μM，对于 GgOse4[sph-3-3H]Cer 为 350 μM，使用 Colo-205 酶）。神经酰胺和鞘氨醇的结构类似物（L-PPMP、L-PDMP、烷基胺和他莫昔芬）在体外抑制癌细胞 CGase 活性。
Ceramide glycanase (CGase) activities have been detected in different human tumor cells (colon, carcinoma Colo-205; neuroblastoma, IMR-32; breast cancer lines, SKBr3 and MCF7). However, the level of enzymatic activity is lower in these cells compared to that present in other mammalian tissues reported before (Basu, M., Kelly, P., Girzadas, M. A., Li, Z., and Basu, S.Methods Enzymol. (in press)). The majority of CGase activity was found in the 100,000gsoluble supernatant fraction isolated from all these cell lines and tissues. Using the soluble enzyme, the requirement for optimum CGase activity was found to be consistent with previous observations found for rat and rabbit tissues (Basu, M., Dastgheib, S., Girzadas, M. A., O'Donnell, P. H., Westervelt, C. W., Li, Z., Inokuchi, J. I., and Basu, S. (1998)Acta Pol. Biochim.42:327). The CGase activities from both Colo-205 and IMR-32 cells are optimum at a protein to detergent ratio of one. All the mammalian CGases, including human cancer cells, show an optimum pH between 5.5 and 5.8 in sodium acetate buffer. The CGase activities from cancer cells are found to be cation-independent; however, mercury, zinc, and copper ions seem to inhibit the enzyme activity substantially in both tumor cells lines. The mercury ion inhibition of CGase activities from all different sources indicates a possible structural homology in the CGase proteins.Radiolabeled substrates, labeled at the sphingosine double bond or at the 3-position of sphingosine without modifying double bond of sphingosine were used in this investigation. Both were active substrates with all enzyme preparations isolated from different cancer cells (apparent Km, 500 μM for nLcOse5[3H-DT]Cer and 350 μM for GgOse4[sph-3-3H]Cer with Colo-205 enzyme). Structural analogues of ceramide and sphingosine (L-PPMP, L-PDMP, alkylamines, and Tamoxifen) inhibited cancer cell CGase activitiesin vitro.