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. 1994 Nov 15;304(Pt 1):105–111. doi: 10.1042/bj3040105

Pteridine biosynthesis and nitric oxide synthase in Physarum polycephalum.

G Werner-Felmayer 1, G Golderer 1, E R Werner 1, P Gröbner 1, H Wachter 1
PMCID: PMC1137459  PMID: 7528004

Abstract

Physarum polycephalum, an acellular slime mould, serves as a model system to study cell-cycle-dependent events since nuclear division is naturally synchronous. This organism was shown to release isoxanthopterin which is structurally related to tetrahydrobiopterin, a cofactor of aromatic amino acid hydroxylases and of nitric oxide synthases (NOSs) (EC 1.14.13.39). Here, we studied Physarum pteridine biosynthesis in more detail and found that high amounts of tetrahydrobiopterin are produced and NOS activity is expressed. Physarum pteridine biosynthesis is peculiar in as much as 7,8-dihydroneopterin aldolase (EC 4.1.2.25), an enzyme of folic acid biosynthesis usually not found in organisms producing tetrahydrobiopterin, is detected in parallel. NOS purified from Physarum depends on NADPH, tetrahydrobiopterin and flavins. Enzyme activity is independent of exogenous Ca2+ and is inhibited by arginine analogues. The purified enzyme (with a molecular mass of 130 kDa) contains tightly bound tetrahydrobiopterin and flavins. During the synchronous cell cycle of Physarum, pteridine biosynthesis increases during S-phase whereas NOS activity peaks during mitosis, drops at telophase and peaks again during early S-phase. Our results characterize Physarum pteridine biosynthesis and NOS and suggest a possible link between NOS activity and mitosis.

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Selected References

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  1. Baek K. J., Thiel B. A., Lucas S., Stuehr D. J. Macrophage nitric oxide synthase subunits. Purification, characterization, and role of prosthetic groups and substrate in regulating their association into a dimeric enzyme. J Biol Chem. 1993 Oct 5;268(28):21120–21129. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Brüne B., Dimmeler S., Molina y Vedia L., Lapetina E. G. Nitric oxide: a signal for ADP-ribosylation of proteins. Life Sci. 1994;54(2):61–70. doi: 10.1016/0024-3205(94)00775-6. [DOI] [PubMed] [Google Scholar]
  4. Bush P. A., Gonzalez N. E., Griscavage J. M., Ignarro L. J. Nitric oxide synthase from cerebellum catalyzes the formation of equimolar quantities of nitric oxide and citrulline from L-arginine. Biochem Biophys Res Commun. 1992 Jun 30;185(3):960–966. doi: 10.1016/0006-291x(92)91720-b. [DOI] [PubMed] [Google Scholar]
  5. Cho H. J., Xie Q. W., Calaycay J., Mumford R. A., Swiderek K. M., Lee T. D., Nathan C. Calmodulin is a subunit of nitric oxide synthase from macrophages. J Exp Med. 1992 Aug 1;176(2):599–604. doi: 10.1084/jem.176.2.599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Clancy R. M., Leszczynska-Piziak J., Abramson S. B. Nitric oxide stimulates the ADP-ribosylation of actin in human neutrophils. Biochem Biophys Res Commun. 1993 Mar 31;191(3):847–852. doi: 10.1006/bbrc.1993.1294. [DOI] [PubMed] [Google Scholar]
  7. Dawson T. M., Steiner J. P., Dawson V. L., Dinerman J. L., Uhl G. R., Snyder S. H. Immunosuppressant FK506 enhances phosphorylation of nitric oxide synthase and protects against glutamate neurotoxicity. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9808–9812. doi: 10.1073/pnas.90.21.9808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Drapier J. C., Hirling H., Wietzerbin J., Kaldy P., Kühn L. C. Biosynthesis of nitric oxide activates iron regulatory factor in macrophages. EMBO J. 1993 Sep;12(9):3643–3649. doi: 10.1002/j.1460-2075.1993.tb06038.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Ferre J., Naylor E. W. Sepiapterin reductase in human amniotic and skin fibroblasts, chorionic villi, and various blood fractions. Clin Chim Acta. 1988 Jun 15;174(3):271–282. doi: 10.1016/0009-8981(88)90053-8. [DOI] [PubMed] [Google Scholar]
  10. Fukushima T., Nixon J. C. Analysis of reduced forms of biopterin in biological tissues and fluids. Anal Biochem. 1980 Feb;102(1):176–188. doi: 10.1016/0003-2697(80)90336-x. [DOI] [PubMed] [Google Scholar]
  11. Green L. C., Wagner D. A., Glogowski J., Skipper P. L., Wishnok J. S., Tannenbaum S. R. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem. 1982 Oct;126(1):131–138. doi: 10.1016/0003-2697(82)90118-x. [DOI] [PubMed] [Google Scholar]
  12. Gross S. S., Levi R. Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem. 1992 Dec 25;267(36):25722–25729. [PubMed] [Google Scholar]
  13. Gross S. S., Stuehr D. J., Aisaka K., Jaffe E. A., Levi R., Griffith O. W. Macrophage and endothelial cell nitric oxide synthesis: cell-type selective inhibition by NG-aminoarginine, NG-nitroarginine and NG-methylarginine. Biochem Biophys Res Commun. 1990 Jul 16;170(1):96–103. doi: 10.1016/0006-291x(90)91245-n. [DOI] [PubMed] [Google Scholar]
  14. Hevel J. M., White K. A., Marletta M. A. Purification of the inducible murine macrophage nitric oxide synthase. Identification as a flavoprotein. J Biol Chem. 1991 Dec 5;266(34):22789–22791. [PubMed] [Google Scholar]
  15. Hölscher C., Rose S. P. An inhibitor of nitric oxide synthesis prevents memory formation in the chick. Neurosci Lett. 1992 Oct 12;145(2):165–167. doi: 10.1016/0304-3940(92)90012-v. [DOI] [PubMed] [Google Scholar]
  16. Kaufman S. New tetrahydrobiopterin-dependent systems. Annu Rev Nutr. 1993;13:261–286. doi: 10.1146/annurev.nu.13.070193.001401. [DOI] [PubMed] [Google Scholar]
  17. Kerler F., Ziegler I., Schmid C., Bacher A. Synthesis of tetrahydrobiopterin in Friend erythroleukemia cells and its modulator effect on cell proliferation. Exp Cell Res. 1990 Aug;189(2):151–156. doi: 10.1016/0014-4827(90)90229-4. [DOI] [PubMed] [Google Scholar]
  18. Klatt P., Schmidt K., Uray G., Mayer B. Multiple catalytic functions of brain nitric oxide synthase. Biochemical characterization, cofactor-requirement, and the role of N omega-hydroxy-L-arginine as an intermediate. J Biol Chem. 1993 Jul 15;268(20):14781–14787. [PubMed] [Google Scholar]
  19. Klein R., Thiery R., Tatischeff I. Dictyopterin, 6-(D-threo-1,2-dihydroxypropyl)-pterin, a new natural isomer of L-biopterin. Isolation from vegetative cells of Dictyostelium discoideum and identification. Eur J Biochem. 1990 Feb 14;187(3):665–669. doi: 10.1111/j.1432-1033.1990.tb15351.x. [DOI] [PubMed] [Google Scholar]
  20. Marletta M. A. Nitric oxide synthase structure and mechanism. J Biol Chem. 1993 Jun 15;268(17):12231–12234. [PubMed] [Google Scholar]
  21. Mayer B., John M., Böhme E. Purification of a Ca2+/calmodulin-dependent nitric oxide synthase from porcine cerebellum. Cofactor-role of tetrahydrobiopterin. FEBS Lett. 1990 Dec 17;277(1-2):215–219. doi: 10.1016/0014-5793(90)80848-d. [DOI] [PubMed] [Google Scholar]
  22. Mayer B., John M., Heinzel B., Werner E. R., Wachter H., Schultz G., Böhme E. Brain nitric oxide synthase is a biopterin- and flavin-containing multi-functional oxido-reductase. FEBS Lett. 1991 Aug 19;288(1-2):187–191. doi: 10.1016/0014-5793(91)81031-3. [DOI] [PubMed] [Google Scholar]
  23. Michel T., Li G. K., Busconi L. Phosphorylation and subcellular translocation of endothelial nitric oxide synthase. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6252–6256. doi: 10.1073/pnas.90.13.6252. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Moncada S., Higgs A. The L-arginine-nitric oxide pathway. N Engl J Med. 1993 Dec 30;329(27):2002–2012. doi: 10.1056/NEJM199312303292706. [DOI] [PubMed] [Google Scholar]
  25. Nathan C. Nitric oxide as a secretory product of mammalian cells. FASEB J. 1992 Sep;6(12):3051–3064. [PubMed] [Google Scholar]
  26. Nichol C. A., Smith G. K., Duch D. S. Biosynthesis and metabolism of tetrahydrobiopterin and molybdopterin. Annu Rev Biochem. 1985;54:729–764. doi: 10.1146/annurev.bi.54.070185.003501. [DOI] [PubMed] [Google Scholar]
  27. Pollock J. S., Förstermann U., Mitchell J. A., Warner T. D., Schmidt H. H., Nakane M., Murad F. Purification and characterization of particulate endothelium-derived relaxing factor synthase from cultured and native bovine aortic endothelial cells. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10480–10484. doi: 10.1073/pnas.88.23.10480. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Rembold H., Heinz G. L-threo-Neopterin, the major pterin in Escherichia coli B. Hoppe Seylers Z Physiol Chem. 1971 Sep;352(9):1271–1272. [PubMed] [Google Scholar]
  29. Ribeiro J. M., Hazzard J. M., Nussenzveig R. H., Champagne D. E., Walker F. A. Reversible binding of nitric oxide by a salivary heme protein from a bloodsucking insect. Science. 1993 Apr 23;260(5107):539–541. doi: 10.1126/science.8386393. [DOI] [PubMed] [Google Scholar]
  30. Sakai N., Kaufman S., Milstein S. Tetrahydrobiopterin is required for cytokine-induced nitric oxide production in a murine macrophage cell line (RAW 264). Mol Pharmacol. 1993 Jan;43(1):6–10. [PubMed] [Google Scholar]
  31. Schmidt H. H., Smith R. M., Nakane M., Murad F. Ca2+/calmodulin-dependent NO synthase type I: a biopteroflavoprotein with Ca2+/calmodulin-independent diaphorase and reductase activities. Biochemistry. 1992 Mar 31;31(12):3243–3249. doi: 10.1021/bi00127a028. [DOI] [PubMed] [Google Scholar]
  32. Stuehr D. J., Cho H. J., Kwon N. S., Weise M. F., Nathan C. F. Purification and characterization of the cytokine-induced macrophage nitric oxide synthase: an FAD- and FMN-containing flavoprotein. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7773–7777. doi: 10.1073/pnas.88.17.7773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Tanaka K., Kaufman S., Milstien S. Tetrahydrobiopterin, the cofactor for aromatic amino acid hydroxylases, is synthesized by and regulates proliferation of erythroid cells. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5864–5867. doi: 10.1073/pnas.86.15.5864. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Viveros O. H., Lee C. L., Abou-Donia M. M., Nixon J. C., Nichol C. A. Biopterin cofactor biosynthesis: independent regulation of GTP cyclohydrolase in adrenal medulla and cortex. Science. 1981 Jul 17;213(4505):349–350. doi: 10.1126/science.7017928. [DOI] [PubMed] [Google Scholar]
  35. Volpe F., Ballantine S. P., Delves C. J. The multifunctional folic acid synthesis fas gene of Pneumocystis carinii encodes dihydroneopterin aldolase, hydroxymethyldihydropterin pyrophosphokinase and dihydropteroate synthase. Eur J Biochem. 1993 Sep 1;216(2):449–458. doi: 10.1111/j.1432-1033.1993.tb18163.x. [DOI] [PubMed] [Google Scholar]
  36. Wachter H., Hausen A., Reider E., Schweiger M. Pteridine excretion from cells as indicator of cell proliferation. Naturwissenschaften. 1980 Dec;67(12):610–611. doi: 10.1007/BF00396550. [DOI] [PubMed] [Google Scholar]
  37. Weiss G., Goossen B., Doppler W., Fuchs D., Pantopoulos K., Werner-Felmayer G., Wachter H., Hentze M. W. Translational regulation via iron-responsive elements by the nitric oxide/NO-synthase pathway. EMBO J. 1993 Sep;12(9):3651–3657. doi: 10.1002/j.1460-2075.1993.tb06039.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Mayer B., Reibnegger G., Weiss G., Wachter H. Ca2+/calmodulin-dependent nitric oxide synthase activity in the human cervix carcinoma cell line ME-180. Biochem J. 1993 Jan 15;289(Pt 2):357–361. doi: 10.1042/bj2890357. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Reibnegger G., Schmidt K., Weiss G., Wachter H. Pteridine biosynthesis in human endothelial cells. Impact on nitric oxide-mediated formation of cyclic GMP. J Biol Chem. 1993 Jan 25;268(3):1842–1846. [PubMed] [Google Scholar]
  40. Werner-Felmayer G., Werner E. R., Fuchs D., Hausen A., Reibnegger G., Wachter H. Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J Exp Med. 1990 Dec 1;172(6):1599–1607. doi: 10.1084/jem.172.6.1599. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Werner E. R., Fuchs D., Hausen A., Reibnegger G., Wachter H. Simultaneous determination of neopterin and creatinine in serum with solid-phase extraction and on-line elution liquid chromatography. Clin Chem. 1987 Nov;33(11):2028–2033. [PubMed] [Google Scholar]
  42. Werner E. R., Werner-Felmayer G., Fuchs D., Hausen A., Reibnegger G., Wachter H. Parallel induction of tetrahydrobiopterin biosynthesis and indoleamine 2,3-dioxygenase activity in human cells and cell lines by interferon-gamma. Biochem J. 1989 Sep 15;262(3):861–866. doi: 10.1042/bj2620861. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Werner E. R., Werner-Felmayer G., Fuchs D., Hausen A., Reibnegger G., Yim J. J., Pfleiderer W., Wachter H. Tetrahydrobiopterin biosynthetic activities in human macrophages, fibroblasts, THP-1, and T 24 cells. GTP-cyclohydrolase I is stimulated by interferon-gamma, and 6-pyruvoyl tetrahydropterin synthase and sepiapterin reductase are constitutively present. J Biol Chem. 1990 Feb 25;265(6):3189–3192. [PubMed] [Google Scholar]
  44. Werner E. R., Werner-Felmayer G., Wachter H. Tetrahydrobiopterin and cytokines. Proc Soc Exp Biol Med. 1993 May;203(1):1–12. doi: 10.3181/00379727-203-43566a. [DOI] [PubMed] [Google Scholar]

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