A continuous microtiter plate assay for screening nucleotide sugar-synthesizing nucleotidyltransferases.
A continuous microtiter plate nucleotidyltransferase substrate screening assay (NUSSA) is described which allows the identification of nucleotide 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 cascade leads to the production of 2 mol NAD per mol PPi. PPi-dependent phosphofructokinase was purified from potato with respect to contaminating enzyme activities which would disturb NUSSA performance. NUSSA allows the quick, simultaneous, and comprehensive check of different sugar 1-phosphate and nucleoside triphosphate substrates using purified pyrophosphorylases or crude extracts of plants, microorganisms, and mammalian tissues. Moreover, NUSSA will assist to evaluate these enzymes for the synthesis of important nucleotide sugars which serve as substrates of glycosyltransferases in carbohydrate syntheses.
Ritter
J E
JE
Institut für Enzymtechnologie, Heinrich-Heine-Universität Düsseldorf im Forschungszentrum Jülich, Germany.
Berlin
C
C
Elling
L
L
D000818:Animals; D011756:Diphosphates; D051599:Glucose-1-Phosphate Adenylyltransferase; D007202:Indicators and Reagents; D008322:Mammals; D009249:NADP / Q000032:analysis; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D009713:Nucleotidyltransferases / Q000032:analysis*; D010732:Phosphofructokinase-1 / Q000032:analysis; D010944:Plants / Q000201:enzymology; D011755:Pyrophosphatases / Q000032:analysis; D011817:Rabbits; D012680:Sensitivity and Specificity; D011198:Solanum tuberosum / Q000201:enzymology; D013379:Substrate Specificity; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase / Q000032:analysis
D011756:Diphosphates; D007202:Indicators and Reagents; D009702:Nucleoside Diphosphate Sugars; D009249:NADP; D010732:Phosphofructokinase-1; D009713:Nucleotidyltransferases; D051599:Glucose-1-Phosphate Adenylyltransferase; D005957:UTP-Glucose-1-Phosphate Uridylyltransferase; D011755:Pyrophosphatases