糖指标的比较可实现通用的高通量糖-1-磷酸核苷酸转移酶检测。
A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay.
作者信息
Moretti Rocco, Thorson Jon S
机构信息
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
出版信息
Anal Biochem. 2008 Jun 15;377(2):251-8. doi: 10.1016/j.ab.2008.03.018. Epub 2008 Mar 15.
A systematic comparison of six sugar indicators for their sensitivity, specificity, cross-reactivity, and suitability in the context of crude lysates revealed para-hydroxybenzoic acid hydrazide (pHBH) to be best suited for application in a plate-based phosphatase-assisted universal sugar-1-phosphate nucleotidyltransferase assay. The addition of a general phosphatase to nucleotidyltransferase reaction aliquots enabled the conversion of remaining sugar-1-phosphate to free sugar, the concentration of which could be rapidly assessed via the pHBH assay. The assay was validated using the model glucose-1-phosphate thymidylyltransferase from Salmonella enterica (RmlA) and compared favorably with a previously reported HPLC assay. This coupled discontinuous assay is quantitative, high throughput, and robust; relies only on commercially available enzymes and reagents; does not require chromatography, specialized detectors (e.g., mass or evaporative light scattering detectors), or radioisotopes; and is capable of detecting less than 5 nmol of sugar-1-phosphate. It is anticipated that this high-throughput assay system will greatly facilitate nucleotidyltransferase mechanistic and directed evolution/engineering studies.
在粗裂解物的背景下，对六种糖指标的敏感性、特异性、交叉反应性和适用性进行了系统比较，结果表明对羟基苯甲酸酰肼（pHBH）最适合用于基于平板的磷酸酶辅助通用糖-1-磷酸核苷酸转移酶测定。向核苷酸转移酶反应等分试样中添加通用磷酸酶可使剩余的糖-1-磷酸转化为游离糖，其浓度可通过pHBH测定快速评估。使用来自肠炎沙门氏菌的模型葡萄糖-1-磷酸胸苷酰转移酶（RmlA）对该测定进行了验证，并与先前报道的HPLC测定相比具有优势。这种偶联的不连续测定是定量的、高通量的且稳健的；仅依赖于市售的酶和试剂；不需要色谱法、专门的检测器（如质谱或蒸发光散射检测器）或放射性同位素；并且能够检测不到5 nmol的糖-1-磷酸。预计这种高通量测定系统将极大地促进核苷酸转移酶的机制和定向进化/工程研究。
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