透明质酸合酶的定量连续测定法。
Quantitative continuous assay for hyaluronan synthase.
作者信息
Krupa Joanne C, Shaya David, Chi Lianli, Linhardt Robert J, Cygler Miroslaw, Withers Stephen G, Mort John S
机构信息
Joint Diseases Laboratory, Shriners Hospital for Children, Montreal, Que., Canada H3G 1A6.
出版信息
Anal Biochem. 2007 Feb 15;361(2):218-25. doi: 10.1016/j.ab.2006.11.011. Epub 2006 Nov 27.
A rapid, continuous, and convenient three-enzyme coupled UV absorption assay was developed to quantitate the glucuronic acid and N-acetylglucosamine transferase activities of hyaluronan synthase from Pasteurella multocida (PmHAS). Activity was measured by coupling the UDP produced from the PmHAS-catalyzed transfer of UDP-GlcNAc and UDP-GlcUA to a hyaluronic acid tetrasaccharide primer with the oxidation of NADH. Using a fluorescently labeled primer, the products were characterized by gel electrophoresis. Our results show that a truncated soluble form of recombinant PmHAS (residues 1-703) can catalyze the glycosyl transfers in a time- and concentration-dependent manner. The assay can be used to determine kinetic parameters, inhibition constants, and mechanistic aspects of this enzyme. In addition, it can be used to quantify PmHAS during purification of the enzyme from culture media.
开发了一种快速、连续且便捷的三酶偶联紫外吸收测定法，用于定量多杀巴斯德菌透明质酸合酶（PmHAS）的葡萄糖醛酸和N-乙酰葡糖胺转移酶活性。通过将PmHAS催化的UDP-GlcNAc和UDP-GlcUA转移至透明质酸四糖引物所产生的UDP与NADH的氧化相偶联来测定活性。使用荧光标记引物，通过凝胶电泳对产物进行表征。我们的结果表明，重组PmHAS的截短可溶性形式（第1至703位氨基酸残基）能够以时间和浓度依赖性方式催化糖基转移。该测定法可用于确定该酶的动力学参数、抑制常数和作用机制。此外，它可用于在从培养基中纯化该酶的过程中对PmHAS进行定量。
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