A CONTINUOUS MICROTITER PLATE ASSAY FOR SCREENING NUCLEOTIDE SUGAR-SYNTHESIZING NUCLEOTIDYLTRANSFERASES

A continuous microtiter plate nucleotidyltransferase substrate screeni ng assay (NUSSA) is described which allows the identification of nucle otide sugar-synthesizing enzyme activities. The assay is accomplished by the determination of the common product of these enzymes PPi with a PPi-dependent phosphofructokinase. A subsequent enzyme reaction casca de leads to the production of 2 mol NAD per mol PPi, PPi-dependent pho sphofructokinase was purified from potato with respect to contaminatin g enzyme activities which would disturb NUSSA performance, NUSSA allow s the quick, simultaneous, and comprehensive check of different sugar 1-phosphate and nucleoside triphosphate substrates using purified pyro phosphorylases or crude extracts of plants, microorganisms, and mammal ian tissues. Moreover, NUSSA will assist to evaluate these enzymes for the synthesis of important nucleotide sugars which serve as substrate s of glycosyltransferases in carbohydrate syntheses. (C) 1996 Academic Press, Inc.