Enzymatic assay procedures that employ high-performance liquid chromatography: competition between phosphoribosyltransferases for a common substrate.
A survey of the phosphoribosyltransferase (PRTase) activities in yeast has been accomplished using reversed-phase high-performance liquid chromatographic assay procedures. The following bases were observed to be utilized during phosphoribosyl pyrophosphate (PRibPP)-dependent nucleotide syntheses: adenine, xanthine, hypoxanthine, guanine, uracil, orotate, nicotinamide, nicotinate and quinolinate. Gradient elution procedures have also been perfected that allow the separation of the two following sets of PRTase assay components: (1) adenosine monophosphate, nicotinate mononucleotide, orotate, adenosine triphosphate, nicotinate, adenosine diphosphate, inosine monophosphate and hypoxanthine, and (2) nicotinate mononucleotide, nicotinamide mononucleotide, adenosine triphosphate, nicotinate, adenosine diphosphate and nicotinamide. Separation 1 has been employed to examine the PRibPP allocation among the hypoxanthine PRTase, orotate PRTase and nicotinate PRTase catalyzed reactions, whereas separation 2 has been employed to define the role that ATP plays in the nicotinamide PRTase-catalyzed reaction along with the allocation of nicotinamide between the reactions catalyzed by nicotinamide PRTase and nicotinamide deamidase.
D000255:Adenosine Triphosphate / Q000378:metabolism; D001667:Binding, Competitive; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism; D009535:Nicotinamidase / Q000378:metabolism; D054409:Nicotinamide Phosphoribosyltransferase; D009705:Nucleosides / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000378:metabolism; D010430:Pentosyltransferases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity
D009705:Nucleosides; D009536:Niacinamide; D000255:Adenosine Triphosphate; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D054409:Nicotinamide Phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D009535:Nicotinamidase