Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells
Catalysis and pH Control by Membrane-associated Carbonic Anhydrase IX in MDA-MB-231 Breast Cancer Cells
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DOI：
10.1074/jbc.m110.188524
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发表时间：
2011-05-06
影响因子：
4.8
通讯作者：
Frost, Susan C.
中科院分区：
文献类型：
--
作者：
Li, Ying;Tu, Chingkuang;Frost, Susan C.
碳酸酐酶 IX (CAIX) 是一种膜结合的肿瘤相关酶，其表达通常被认为是缺氧的标志物，是大多数癌症患者预后不良的指标，并且与肿瘤微环境的酸化有关。在这里，我们首次描述了 MDA-MB-231 乳腺癌细胞中天然 CAIX 的催化特性，这些细胞表现出缺氧诱导的 CAIX 表达。利用膜入口质谱法测量的 O-18 交换，我们确定了膜鬼和完整细胞的催化活性。面外碳酸酐酶活性随着缺氧而增加，而非渗透性磺酰胺 CA 抑制剂会抑制该活性。磺酰胺抑制剂的抑制作用对复氧不敏感。完整细胞中的 CAIX 活性随着 pH 值降低而增加。来自膜鬼影的数据表明，pH 值降低时活性的增加主要是由于脱水反应的增加。此外，膜鬼影中 CAIX 的动力学常数与我们之前对纯化、重组、截短形式的测量非常相似。因此，CAIX的活性不受蛋白聚糖延伸或膜环境的影响。这些活性是在所有 CO2 物种的总浓度为 25 mM 且接近化学平衡（接近生理细胞外环境的条件）下测量的。我们的数据表明 CAIX 特别适合将细胞外 pH 值维持在有利于肿瘤细胞生存的值。
Carbonic anhydrase IX (CAIX) is a membrane-bound, tumor-related enzyme whose expression is often considered a marker for hypoxia, an indicator of poor prognosis in the majority of cancer patients, and is associated with acidification of the tumor microenvironment. Here, we describe for the first time the catalytic properties of native CAIX in MDA-MB-231 breast cancer cells that exhibit hypoxia-inducible CAIX expression. Using O-18 exchange measured by membrane inlet mass spectrometry, we determined catalytic activity in membrane ghosts and intact cells. Exofacial carbonic anhydrase activity increases with exposure to hypoxia, an activity which is suppressed by impermeant sulfonamide CA inhibitors. Inhibition by sulfonamide inhibitors is not sensitive to reoxygenation. CAIX activity in intact cells increases in response to reduced pH. Data from membrane ghosts show that the increase in activity at reduced pH is largely due to an increase in the dehydration reaction. In addition, the kinetic constants of CAIX in membrane ghosts are very similar to our previous measurements for purified, recombinant, truncated forms. Hence, the activity of CAIX is not affected by the proteoglycan extension or membrane environment. These activities were measured at a total concentration for all CO2 species at 25 mM and close to chemical equilibrium, conditions which approximate the physiological extracellular environment. Our data suggest that CAIX is particularly well suited to maintain the extracellular pH at a value that favors the survival fitness of tumor cells.