This project compared thermodynamic data obtained in vitro for the purine salvage reactions to the measured steady-state levels of purine and pyrimidine nucleobases, nucleosides, nucleotides, and related cofactor compounds in vivo. (The hypothesis is that the purine salvage enzymes catalyze reactions that approach near-equilibrium, in vivo, and thus that rates of synthesis of the necessary cofactors such as ribose 1-phosphate (R1-P), phosphoribosylpyrophosphate (PRPP), inorganic phosphate (Pi), and inorganic pyrophosphate (PPi), could profoundly influence the relative amounts of free bases and nucleosides available for nucleotide and urate production.) Current methods for the extraction and quantitation of purine and pyrimidine nucleobases and nucleosides as well as for R1-P and PRPP are suspect and have led to confusion and posibly misinterpretation of scientific data. Therefore, we began by attempting to improve on current methods for measuring the aforementioned metabolites in liver extracts. A superior high-performance liquid chromatograhic procedure was developed for measuring purine and pyrimidine nucleo bases and nucleosides in rat hepatocytes. Progress was also made on improving techniques for the extraction and quantitation of R1-P and PRPP. The apparent equilibrium constants for the purine salvage reactions catalyzed by purine nucleoside phosphorylase were completed as well as for the ancillary reactions, phosphoribomutase and 5' nucleotidase. The equilibrium constant for the phosphoribosyltransferases will be completed once PRPP can be measured. Once these in vivo measurements are made it can be determined whether the salvage reactions approach near-equilibrium in vivo and, if so, whether it will be possible to assess the influence of fluctuations in the magnitude of the cofactor couples PRPP/PPi and R1-P/Pi on the rates of nucleotide, nucleic acid, and uric acid synthesis in a variety of cell types.
