Gosselin S, Alhussaini M, Streiff M B, Takabayashi K, Palcic M M
Department of Chemistry, University of Alberta, Edmonton, Canada.
Anal Biochem. 1994 Jul;220(1):92-7. doi: 10.1006/abio.1994.1303.
This paper describes a continuous spectrophotometric assay for glycosyltransferases. In this assay, a nucleotide diphosphate is coupled to NADH oxidation via pyruvate kinase and lactate dehydrogenase. The nucleotide diphosphate is produced either directly during the glycosyltransferase mediated reaction, or indirectly by the production of a nucleotide monophosphate during the glycosyltransferase mediated reaction, and subsequent conversion of the nucleotide monophosphate to nucleotide diphosphate using nucleoside monophosphate kinase. Using this assay, kinetic parameters for fucosyl-, sialyl-, and N-acetylglucosaminyltransferases were determined. The assay not only allows continual monitoring of the enzymatic reaction, but is rapid and allows the processing of 96 samples at once since it is performed in 96-well microtiter plates. In addition, the procedure provides a means of monitoring the activity of these enzymes using sugar-nucleotide donor analogs, where radiochemical procedures cannot be used.
本文描述了一种用于糖基转移酶的连续分光光度测定法。在该测定法中，核苷二磷酸通过丙酮酸激酶和乳酸脱氢酶与NADH氧化偶联。核苷二磷酸要么在糖基转移酶介导的反应中直接产生，要么在糖基转移酶介导的反应中通过产生核苷一磷酸间接产生，随后使用核苷单磷酸激酶将核苷一磷酸转化为核苷二磷酸。使用该测定法，确定了岩藻糖基转移酶、唾液酸基转移酶和N-乙酰葡糖胺基转移酶的动力学参数。该测定法不仅允许对酶促反应进行连续监测，而且速度快，由于它是在96孔微量滴定板中进行的，因此可以一次处理96个样品。此外，该方法提供了一种使用糖核苷酸供体类似物监测这些酶活性的手段，而在无法使用放射化学方法的情况下。
