Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1982 Feb;79(4):1037–1041. doi: 10.1073/pnas.79.4.1037

Isolation and partial characterization of a 5'-nucleotidase specific for orotidine-5'-monophosphate.

M H El Kouni, S Cha
PMCID: PMC345894  PMID: 6280163

Abstract

A previously unknown 5'nucleotidase (5'-ribonucleotide phosphohydrolase, EC 3.1.3.5) (5'-Nase) specific for orotidine 5'-monophosphate (OMP) hs been discovered. This enzyme orotidine 5'-monophosphate phosphohydrolase (OMPase), was isolated from mouse liver microsomes as a separate entity from the nonspecific 5'-Nase. OMPase was partially purified and is shown to cleave OMP to orotidine and inorganic phosphate. The enzyme has negligible activity towards UMP, CMP, dTMP, AMP, IMP, GMP, XMP, 6-azauridine 5'-monophosphate, 1-beta-D-ribofuranosylbarbituric acid 5'-monophosphate (BMF), 2'-UMP, 3'-UMP, 2'-AMP, 3'-AMP, ribose 5-phosphate and beta-glycerophosphate, all of which--with the exception of the 2' or 3' monophosphates, ribose 5'-phosphate, and beta-glycerophosphate--are substrates for 5'-Nase. Both enzymes are inhibited by NaF, but only OMPase is inhibited by SF reagents. OMPase is not inhibited by orotidine, orotate, BMP, concanavalin A, or tetramisole (an alkaline phosphatase inhibitor). OMPase had a Mr 53,000, Km value of 1 mM for OMP, and Vmax value of 49 nmol/min . mg of protein at the present stage of purification. OMPase activity has also been detected in various mammalian tissues including normal human tissues, human tumor xenografts, lymphocytes, and rat liver. OMPase may be responsible, in part, for the low levels of intracellular "free" OMP and for orotidine accumulation in cells treated with 6-azauridine and patients suffering from aortic aciduria.

Full text

PDF
1037

Selected References

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

  1. Dornand J., Bonnafous J. C., Mani J. C. Purification and properties of 5'-nucleotidase from lymphocyte plasma membranes. Eur J Biochem. 1978 Jul 3;87(3):459–465. doi: 10.1111/j.1432-1033.1978.tb12396.x. [DOI] [PubMed] [Google Scholar]
  2. Evans W. H., Gurd J. W. Properties of a 5'-nucleotidase purified from mouse liver plasma membranes. Biochem J. 1973 May;133(1):189–199. doi: 10.1042/bj1330189. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Fox R. M., Tripp E. H., Piddington S. K., Tattersall M. H. Sensitivity of leukemic human null lymphocytes to deoxynucleosides. Cancer Res. 1980 Sep;40(9):3383–3386. [PubMed] [Google Scholar]
  4. Hayman M. J., Crumpton M. J. Isolation of glycoproteins from pig lymphocyte plasma membrane using Lens culinaris phytohemagglutinin. Biochem Biophys Res Commun. 1972 May 26;47(4):923–930. doi: 10.1016/0006-291x(72)90581-5. [DOI] [PubMed] [Google Scholar]
  5. Holloway P. W. A simple procedure for removal of Triton X-100 from protein samples. Anal Biochem. 1973 May;53(1):304–308. doi: 10.1016/0003-2697(73)90436-3. [DOI] [PubMed] [Google Scholar]
  6. Janeway C. M., Cha S. Effects of 6-azauridine on nucleotides, orotic acid, and orotidine in L5178Y mouse lymphoma cells in vitro. Cancer Res. 1977 Dec;37(12):4382–4388. [PubMed] [Google Scholar]
  7. Jones M. E. Pyrimidine nucleotide biosynthesis in animals: genes, enzymes, and regulation of UMP biosynthesis. Annu Rev Biochem. 1980;49:253–279. doi: 10.1146/annurev.bi.49.070180.001345. [DOI] [PubMed] [Google Scholar]
  8. Lee M. H., Huang Y. M., Agrawal K. C., Sartorelli A. C. Inhibitors of alkaline phosphatase of Sarcoma 180/TG. Biochem Pharmacol. 1975 Jun 15;24(11-12):1175–1178. [PubMed] [Google Scholar]
  9. Levine H. L., Brody R. S., Westheimer F. H. Inhibition of orotidine-5'-phosphate decarboxylase by 1-(5'-phospho-beta-d-ribofuranosyl)barbituric acid, 6-azauridine 5'-phosphate, and uridine 5'-phosphate. Biochemistry. 1980 Oct 28;19(22):4993–4999. doi: 10.1021/bi00563a010. [DOI] [PubMed] [Google Scholar]
  10. Paglia D. E., Valentine W. N. Characteristics of a pyrimidine-specific 5'-nucleotidase in human erythrocytes. J Biol Chem. 1975 Oct 25;250(20):7973–7979. [PubMed] [Google Scholar]
  11. Potvin B. W., Stern H. J., May S. R., Lam G. F., Krooth R. S. Inhibition by barbituric acid and its derivatives of the enzymes in rat brain which participate in the synthesis of pyrimidine ribotides. Biochem Pharmacol. 1978 Mar 1;27(5):655–665. doi: 10.1016/0006-2952(78)90501-4. [DOI] [PubMed] [Google Scholar]
  12. Scholar E. M., Calabresi P. Identification of the enzymatic pathways of nucleotide metabolism in human lymphocytes and leukemia cells. Cancer Res. 1973 Jan;33(1):94–103. [PubMed] [Google Scholar]
  13. Slavik M., Kartner N., Riordan J. R. Lectin-induced inhibition of plasma membrane 5'-nucleotidase: sensitivity of purified enzyme. Biochem Biophys Res Commun. 1977 Mar 21;75(2):342–349. doi: 10.1016/0006-291x(77)91048-8. [DOI] [PubMed] [Google Scholar]
  14. Tax W. J., Veerkamp J. H., Schretlen E. D. Pyrimidine metabolism in erythrocytes of the newborn. Biol Neonate. 1979;35(3-4):121–125. doi: 10.1159/000241162. [DOI] [PubMed] [Google Scholar]
  15. Traut T. W. Significance of the enzyme complex that synthesizes UMP in Ehrlich ascites cells. Arch Biochem Biophys. 1980 Apr 1;200(2):590–594. doi: 10.1016/0003-9861(80)90391-4. [DOI] [PubMed] [Google Scholar]
  16. Valentine W. N., Fink K., Paglia D. E., Harris S. R., Adams W. S. Hereditary hemolytic anemia with human erythrocyte pyrimidine 5'-nucleotidase deficiency. J Clin Invest. 1974 Oct;54(4):866–879. doi: 10.1172/JCI107826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Van Belle H. Kinetics and inhibition of alkaline phosphatases from canine tissues. Biochim Biophys Acta. 1972 Nov 10;289(1):158–168. doi: 10.1016/0005-2744(72)90118-0. [DOI] [PubMed] [Google Scholar]
  18. el Kouni M. H., Cha S. A simple radioisotopic assay for nucleoside kinases employing alumina for separation of nucleosides and nucleotides. Anal Biochem. 1981 Feb;111(1):67–71. doi: 10.1016/0003-2697(81)90229-3. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES