Enzymatic coupled assay procedures that employ high-performance liquid chromatography: the synthesis of orotidylate from ribose.
High-performance liquid chromatographic assay procedures have been designed to monitor the catalytic activities of ribokinase and phosphoribosyl alpha-1-pyrophosphate (PRibPP) synthetase. These methods are only of qualitative value, when crude protein extracts are to be examined, because of the presence of myokinase. However, the product of the PRibPP synthetase reaction, can be detected quantitatively even in crude protein extracts through the addition of two enzymes (orotate phosphoribosyltransferase and inorganic pyrophosphatase) that catalyze the conversion of PRibPP into a spectroscopically detectable nucleotide product (orotidylate).
D001426:Bacterial Proteins / Q000378:metabolism; D055598:Chemical Phenomena; D002621:Chemistry; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D010428:Pentosephosphates / Q000378:metabolism; D010754:Phosphoribosyl Pyrophosphate; D010770:Phosphotransferases / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor); D012266:Ribose / Q000378:metabolism*; D012268:Ribose-Phosphate Pyrophosphokinase / Q000378:metabolism; D012486:Salmonella typhimurium / Q000201:enzymology; D014500:Uracil Nucleotides / Q000096:biosynthesis*; D014542:Uridine Monophosphate / Q000031:analogs & derivatives / Q000096:biosynthesis*
D001426:Bacterial Proteins; D010428:Pentosephosphates; D014500:Uracil Nucleotides; C005417:orotidylic acid; D012266:Ribose; D010754:Phosphoribosyl Pyrophosphate; D014542:Uridine Monophosphate; D010770:Phosphotransferases; D017853:Phosphotransferases (Alcohol Group Acceptor); C022534:ribokinase; D012268:Ribose-Phosphate Pyrophosphokinase