Abstract
A high-pressure liquid chromatography (hplc) procedure has been designed to monitor the reaction catalyzed by nicotinate phosphoribosyltransferase (N-PRTase). Two substrates (ATP and nicotinate) and two products of this reaction (nicotinate mononucleotide and ADP) can be resolved at pH 8 on a Waters μBondapak C 18 column, and the concentration of each reactant in the isocratic eluant (25 m m (NH 4) 3PO 4) can be determined spectroscopically at 254 nm. This separation method allows an analysis of the disappearance of substrates and the appearance of products simultaneously during the time course of the enzyme-catalyzed reaction. We have employed this new N-PRTase assay system: (1) to prove that a 1:1 stoichiometry exists between the enzymatic ATP hydrolysis and nicotinate mononucleotide formation and (2) to reveal for the first time an inhibition of the overall reaction by phosphoribosyl α-1-pyrophosphate at relatively low ATP concentrations.
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