Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1983 Oct;156(1):198–204. doi: 10.1128/jb.156.1.198-204.1983

Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.

R S McIvor, R M Wohlhueter, P G Plagemann
PMCID: PMC215070  PMID: 6619095

Abstract

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-14C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (KmUrd = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.

Full text

PDF
204

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Beck C. F., Ingraham J. L., Neuhard J., Thomassen E. Metabolism of pyrimidines and pyrimidine nucleosides by Salmonella typhimurium. J Bacteriol. 1972 Apr;110(1):219–228. doi: 10.1128/jb.110.1.219-228.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bose R., Yamada E. W. Uridine phosphorylase, molecular properties and mechanism of catalysis. Biochemistry. 1974 May 7;13(10):2051–2056. doi: 10.1021/bi00707a008. [DOI] [PubMed] [Google Scholar]
  3. FRIEDKIN M., ROBERTS D. The enzymatic synthesis of nucleosides. I. Thymidine phosphorylase in mammalian tissue. J Biol Chem. 1954 Mar;207(1):245–256. [PubMed] [Google Scholar]
  4. Gotto A. M., Belkhode M. L., Touster O. Stimulatory effects of inosine and deoxyinosine on the incorporation of uracil-2-14-C, 5-fluorouracil-2-14-C, and 5-bromouracil-2-14-C into nucleic acids by Ehrlich ascites tumor cells in vitro. Cancer Res. 1969 Apr;29(4):807–811. [PubMed] [Google Scholar]
  5. HAKALA M. T., HOLLAND J. F., HOROSZEWICZ J. S. Change in pyrimidine deoxyribonucleoside metabolism in cell culture caused by Mycoplasma (PPLO) contamination. Biochem Biophys Res Commun. 1963 Jun 20;11:466–471. doi: 10.1016/0006-291x(63)90094-9. [DOI] [PubMed] [Google Scholar]
  6. Hamet M., Bonissol C., Cartier P. Enzymatic activities on purine pyrimidine metabolism in nine mycoplasma species contaminating cell cultures. Clin Chim Acta. 1980 Apr 11;103(1):15–22. doi: 10.1016/0009-8981(80)90225-9. [DOI] [PubMed] [Google Scholar]
  7. Itzhaki S. Regulation of uracil utilization for RNA synthesis in Ehrlich ascites tumour cells by glucose metabolism. Life Sci II. 1972 Jul 8;11(13):649–655. doi: 10.1016/0024-3205(72)90013-6. [DOI] [PubMed] [Google Scholar]
  8. Kraut A., Yamada E. W. Cytoplsmic uridine phosphorylase of rat liver. Characterization and kinetics. J Biol Chem. 1971 Apr 10;246(7):2021–2030. [PubMed] [Google Scholar]
  9. Krenitsky T. A. Uridine phosphorylase from Escherichia coli. Kinetic properties and mechanism. Biochim Biophys Acta. 1976 Apr 8;429(2):352–358. doi: 10.1016/0005-2744(76)90283-7. [DOI] [PubMed] [Google Scholar]
  10. Leer J. C., Hammer-Jespersen K., Schwartz M. Uridine phosphorylase from Escherichia coli. Physical and chemical characterization. Eur J Biochem. 1977 May 2;75(1):217–224. doi: 10.1111/j.1432-1033.1977.tb11520.x. [DOI] [PubMed] [Google Scholar]
  11. Levine E. M. Mycoplasma contamination of animal cell cultures: a simple, rapid detection method. Exp Cell Res. 1972 Sep;74(1):99–109. doi: 10.1016/0014-4827(72)90484-3. [DOI] [PubMed] [Google Scholar]
  12. McIvor R. S., Wohlhueter R. M., Plagemann P. G. An improved enzymatic radioassay for ribose 1-phosphate. Anal Biochem. 1982 Nov 15;127(1):150–154. doi: 10.1016/0003-2697(82)90157-9. [DOI] [PubMed] [Google Scholar]
  13. McIvor R. S., Wohlhueter R. M., Plagemann P. G. Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties. J Bacteriol. 1983 Oct;156(1):192–197. doi: 10.1128/jb.156.1.192-197.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Milman G., Anton D. L., Weber J. L. Chinese hamster purine-nucleoside phosphorylase: purification, structural, and catalytic properties. Biochemistry. 1976 Nov 16;15(23):4967–4973. doi: 10.1021/bi00668a004. [DOI] [PubMed] [Google Scholar]
  15. Mitchell A., Finch L. R. Enzymes of pyrimidine metabolism in Mycoplasma mycoides subsp. mycoides. J Bacteriol. 1979 Mar;137(3):1073–1080. doi: 10.1128/jb.137.3.1073-1080.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Mitchell A., Finch L. R. Pathways of nucleotide biosynthesis in Mycoplasma mycoides subsp. mycoides. J Bacteriol. 1977 Jun;130(3):1047–1054. doi: 10.1128/jb.130.3.1047-1054.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. O'Donovan G. A., Neuhard J. Pyrimidine metabolism in microorganisms. Bacteriol Rev. 1970 Sep;34(3):278–343. doi: 10.1128/br.34.3.278-343.1970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. PAEGE L. M., SCHLENK F. Pyrimidine riboside metabolism. Arch Biochem. 1950 Oct;28(3):348–358. [PubMed] [Google Scholar]
  19. PONTIS H., DEGERSTEDT G., REICHARD P. Uridine and deoxyuridine phosphorylases from Ehrlich ascites tumor. Biochim Biophys Acta. 1961 Jul 22;51:138–147. doi: 10.1016/0006-3002(61)91024-1. [DOI] [PubMed] [Google Scholar]
  20. Plagemann P. G., Wohlhueter R. M., Erbe J. Facilitated transport of inosine and uridine in cultured mammalian cells is independent of nucleoside phosphorylases. Biochim Biophys Acta. 1981 Jan 22;640(2):448–462. doi: 10.1016/0005-2736(81)90470-3. [DOI] [PubMed] [Google Scholar]
  21. Wohlhueter R. M. Hypoxanthine phosphoribosyltransferase activity in normal, developing, and neoplastic tissues of the rat. Eur J Cancer. 1975 Jul;11(7):463–472. doi: 10.1016/0014-2964(75)90147-4. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES