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首页> 美国卫生研究院文献>Protein Science : A Publication of the Protein Society >Interaction of phlorizin a potent inhibitor of the Na+/D-glucose cotransporter with the NADPH-binding site of mammalian catalases.
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Interaction of phlorizin a potent inhibitor of the Na+/D-glucose cotransporter with the NADPH-binding site of mammalian catalases.

机译：Na + / D-葡萄糖共转运蛋白的有效抑制剂Phlorizin与哺乳动物过氧化氢酶的NADPH结合位点的相互作用。

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摘要

Phlorizin is a reversible inhibitor of the renal and small intestinal Na+/D-glucose cotransporter. In an attempt to purify the Na+/D-glucose cotransporter from a pig kidney brush border membrane fraction, we used an Affi-Gel affinity chromatography column to which 3-aminophlorizin had been coupled. A protein, composed according to crosslinking experiments of at least 3 subunits of molecular weight 60 kDa, was found to bind specifically to the phlorizin column. This protein was subsequently identified as catalase by sequence homology of three of its tryptic fragments to the sequence of several mammalian catalases as well as by its enzymatic activity. Although bovine liver catalase was bound tightly to the affinity matrix, phlorizin had no effect on the ability of the enzyme to degrade H2O2. In contrast, the Aspergillus niger and Neurospora crassa catalases did not bind to the phlorizin column. This difference may be related to the fact that mammalian catalases, but not the fungal catalases, contain an NADPH binding site with a yet unknown function. Interestingly, bovine liver catalase could be eluted with 50 microM NADPH from phlorizin columns. Irradiation in the presence of [3H]4-azidophlorizin allowed photolabeling of bovine liver catalase, which was prevented by the presence of 10 microM NADPH. After digestion of photolabeled catalase with chymotrypsin, a radioactive peptide was detected that was absent in catalase protected with NADPH. Docking simulations suggested that phlorizin can bind to the NADPH binding site with high affinity.
机译：Phlorizin是肾脏和小肠Na + / D-葡萄糖共转运蛋白的可逆抑制剂。为了从猪肾刷状边缘膜级分纯化Na + / D-葡萄糖共转运蛋白，我们使用了Affi-Gel亲和色谱柱，该色谱柱已与3-aminophlorizin偶联。根据交联实验，由至少三个分子量为60 kDa的亚基组成的蛋白质被发现与Phlorizin柱特异性结合。随后通过其三个胰蛋白酶片段与几种哺乳动物过氧化氢酶序列的序列同源性及其酶促活性将该蛋白鉴定为过氧化氢酶。尽管牛肝过氧化氢酶与亲和基质紧密结合，但是phlorizin对酶降解H2O2的能力没有影响。相比之下，黑曲霉和克雷索氏菌的过氧化氢酶不结合到phlorizin柱上。这种差异可能与以下事实有关：哺乳动物的过氧化氢酶（而非真菌的过氧化氢酶）包含具有未知功能的NADPH结合位点。有趣的是，牛肝过氧化氢酶可以用50 microM NADPH从phlorizin柱上洗脱。在[3H] 4-叠氮唑嗪的存在下进行辐照可以对牛肝过氧化氢酶进行光标记，这可以通过存在10 microM NADPH来防止。用胰凝乳蛋白酶消化光标记的过氧化氢酶后，检测到在用NADPH保护的过氧化氢酶中不存在的放射性肽。对接模拟表明，phlorizin可以高亲和力结合到NADPH结合位点。

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