Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase.
Biochemical and pathophysiological characterization of Helicobacter pylori asparaginase.
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幽门螺杆菌天冬酰胺酶的生化和病理生理学特征。
DOI：
10.1111/j.1348-0421.2012.00459.x
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发表时间：
2012
影响因子：
2.6
通讯作者：
Shibayama K
中科院分区：
文献类型：
--
作者：
Nakayama K;Nakamura H;Koga M;Koibuchi T;Fujii T;Miura T;Iwamoto A;Kawana-Tachikawa A.;Shibayama K
我们读了斯科蒂等人的信。(1)，他对我们关于幽门螺杆菌天冬酰胺酶特性的论文进行了评论（2）。针对我们关于他们的重组天冬酰胺酶可能没有正确制备的声明，他们回应说，基于凝胶过滤和圆二色谱分析以及四级结构预测，他们的蛋白质具有完美的活性。我们同意他们的重组蛋白是可溶性的和多聚体的;然而，基于他们的论文（3）中描述的结果，我们认为重组蛋白不能准确模拟实际的H。幽门螺杆菌天冬酰胺酶。作者报道，0.021 U/mL的H. pylori天冬酰胺酶。pylori培养基抑制HDF细胞的细胞周期（表2;图2A（3））。然而，即使在0.5 U/mL（相当于16 µg/mL）的浓度下，其重组蛋白也不会抑制这些细胞的细胞周期（图3A（3））。这种差异表明重组蛋白与天然蛋白有很大的不同。pylori天冬酰胺酶。我们通过使用高灵敏度和准确的高效液相色谱法直接测量产生的天冬氨酸来确定天冬酰胺的Vmax和Km。仅考虑反应的线性阶段;如我们发表的论文（2）中所述，测量进行了长达30 s。与此相反，由Scotti等人使用的Nessler试剂的氨的分光光度测量。(4)是相对近似的。将氨定量到1 µM以下是不切实际的，这是这种情况下所需的浓度。总之，当分析难以纯化的蛋白质时，重组构建体可能是有帮助的。然而，在这方面，
We read the letter by Scotti et al.(1), who commented on our paper about the characterization of Helicobacter pylori asparaginase (2). In response to our statement that their recombinant asparaginase may not be prepared properly, they responded that their protein was perfectly active based on gel filtration and circular dichroism analyses, and a quaternary structure prediction. We do agree that their recombinant protein was soluble and multimeric; however, based on the results described in their paper (3), we believe that the recombinant protein does not accurately mimic the actual H. pylori asparaginase. The authors reported that 0.021 U/mL of H. pylori asparaginase extracted from H. pylori culture medium inhibited the cell cycle in HDF cells (table 2; figure 2A (3)). However, their recombinant protein did not inhibit the cell cycle in these cells, even at a concentration of 0.5 U/mL, which corresponds to 16 µg/mL (fig. 3A (3)). This difference indicates that the recombinant protein was substantially different from the native protein.We used the native H. pylori asparaginase for all assays. We determined the Vmax and Km of asparagine by directly measuring aspartate produced using highly sensitive and accurate high-performance liquid chromatography. Only the linear phase of the reaction was considered; the measurements were carried out for up to 30 s, as described in our published paper (2). In contrast, the spectrophotometric measurement of ammonia by Nessler’s reagent used by Scotti et al.(4) is relatively approximate. It is not practical to quantify ammonia below 1 µM, which is the required concentration in this case. In summary, when analyzing difficult-to-purify proteins, recombinant constructs can be helpful. However,