A Continuous Microtiter Plate Assay for Screening Nucleotide Sugar-Synthesizing Nucleotidyltransferases

Authors: Ritter J.E.; Berlin C.; Elling L.

Source: Analytical Biochemistry, Volume 234, Number 1, February 1996 , pp. 74-82(9)

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Abstract:

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 PP i with a PP i -dependent phosphofructokinase. A subsequent enzyme reaction cascade leads to the production of 2 mol NAD per mol PP i . PP i -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.

Language: English

Document Type: Research article

Affiliations: Institut fur Enzymtechnologie der Heinrich-Heine-Universitat Dusseldorf im Forschungszentrum Julich, Julich, D-52404, Germany

Publication date: 1996-02-01