A CONTINUOUS SPECTROPHOTOMETRIC ASSAY FOR GLYCOSYLTRANSFERASES

This paper describes a continuous spectrophotometric assay for glycosy ltransferases. In this assay, a nucleotide diphosphate is coupled to N ADH oxidation via pyruvate kinase and lactate dehydrogenase. The nucle otide diphosphate is produced either directly during the glycosyltrans ferase mediated reaction, or indirectly by the production of a nucleot ide monophosphate during the glycosyltransferase mediated reaction, an d subsequent conversion of the nucleotide monophosphate to nucleotide diphosphate using nucleoside monophosphate kinase. Using this assay, k inetic parameters for fucosyl-, sialyl-, and N-acetylglucosaminyltrans ferases were determined. The assay not only allows continual monitorin g 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 activi ty of these enzymes using sugar-nucleotide donor analogs, where radioc hemical procedures cannot be used. (C) 1994 Academic Press, Inc.