Skip to main content
PMC home page
Biochemical Journal logoLink to Biochemical Journal
. 2002 Nov 1;367(Pt 3):665–675. doi: 10.1042/BJ20020720

Identification and characterization of a novel flavin-containing spermine oxidase of mammalian cell origin.

Slavoljub Vujcic 1, Paula Diegelman 1, Cyrus J Bacchi 1, Debora L Kramer 1, Carl W Porter 1
PMCID: PMC1222929  PMID: 12141946

Abstract

During polyamine catabolism, spermine and spermidine are first acetylated by spermidine/spermine N(1)-acetyltransferase (SSAT) and subsequently oxidized by polyamine oxidase (PAO) to produce spermidine and putrescine, respectively. In attempting to clone the PAO involved in this back-conversion pathway, we encountered an oxidase that preferentially cleaves spermine in the absence of prior acetylation by SSAT. A BLAST search using maize PAO sequences identified homologous mammalian cDNAs derived from human hepatoma and mouse mammary carcinoma: the encoded proteins differed by 20 amino acids. When either cDNA was transiently transfected into HEK-293 cells, intracellular spermine pools decreased by 75% while spermidine and N (1)-acetylspermidine pools increased, suggesting that spermine was selectively and directly oxidized by the enzyme. Substrate specificity using lysates of oxidase-transfected HEK-293 cells revealed that the newly identified oxidase strongly favoured spermine over N (1)-acetylspermine and that it failed to act on N (1)-acetylspermidine, spermidine or the preferred PAO substrate, N (1), N (12)-diacetylspermine. The PAO inhibitor, MDL-72,527, only partially blocked oxidation of spermine while a previously reported PAO substrate, N (1)-( n -octanesulphonyl)spermine, potently inhibited the reaction. Overall, the data indicate that the enzyme represents a novel mammalian oxidase which, on the basis of substrate specificity, we have designated spermine oxidase in order to distinguish it from the PAO involved in polyamine back-conversion. The identification of an enzyme capable of directly oxidizing spermine to spermidine has important implications for understanding polyamine homoeostasis and for interpreting metabolic and cellular responses to clinically relevant polyamine analogues and inhibitors.

Full Text

The Full Text of this article is available as a PDF (250.2 KB).

Selected References

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

  1. Alhonen L., Karppinen A., Uusi-Oukari M., Vujcic S., Korhonen V. P., Halmekytö M., Kramer D. L., Hines R., Jänne J., Porter C. W. Correlation of polyamine and growth responses to N1,N11-diethylnorspermine in primary fetal fibroblasts derived from transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem. 1998 Jan 23;273(4):1964–1969. doi: 10.1074/jbc.273.4.1964. [DOI] [PubMed] [Google Scholar]
  2. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  3. Bacchi C. J., Goldberg B., Garofalo-Hannan J., Rattendi D., Lyte P., Yarlett N. Fate of soluble methionine in African trypanosomes: effects of metabolic inhibitors. Biochem J. 1995 Aug 1;309(Pt 3):737–743. doi: 10.1042/bj3090737. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bacchi C. J., Lane S., Weiss L. M., Yarlett N., Takvorian P., Cali A., Wittner M. Polyamine synthesis and interconversion by the Microsporidian Encephalitozoon cuniculi. J Eukaryot Microbiol. 2001 May-Jun;48(3):374–381. doi: 10.1111/j.1550-7408.2001.tb00327.x. [DOI] [PubMed] [Google Scholar]
  5. Bateman A., Birney E., Durbin R., Eddy S. R., Finn R. D., Sonnhammer E. L. Pfam 3.1: 1313 multiple alignments and profile HMMs match the majority of proteins. Nucleic Acids Res. 1999 Jan 1;27(1):260–262. doi: 10.1093/nar/27.1.260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bey P., Bolkenius F. N., Seiler N., Casara P. N-2,3-Butadienyl-1,4-butanediamine derivatives: potent irreversible inactivators of mammalian polyamine oxidase. J Med Chem. 1985 Jan;28(1):1–2. doi: 10.1021/jm00379a001. [DOI] [PubMed] [Google Scholar]
  7. Bitonti A. J., Dumont J. A., Bush T. L., Stemerick D. M., Edwards M. L., McCann P. P. Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase. J Biol Chem. 1990 Jan 5;265(1):382–388. [PubMed] [Google Scholar]
  8. Bolkenius F. N., Bey P., Seiler N. Specific inhibition of polyamine oxidase in vivo is a method for the elucidation of its physiological role. Biochim Biophys Acta. 1985 Jan 28;838(1):69–76. doi: 10.1016/0304-4165(85)90251-x. [DOI] [PubMed] [Google Scholar]
  9. Bolkenius F. N., Seiler N. Acetylderivatives as intermediates in polyamine catabolism. Int J Biochem. 1981;13(3):287–292. doi: 10.1016/0020-711x(81)90080-x. [DOI] [PubMed] [Google Scholar]
  10. Bolkenius F. N., Seiler N. New substrates of polyamine oxidase. Dealkylation of N-alkyl-alpha, omega-diamines. Biol Chem Hoppe Seyler. 1989 Jun;370(6):525–531. doi: 10.1515/bchm3.1989.370.1.525. [DOI] [PubMed] [Google Scholar]
  11. Casero R. A., Jr, Celano P., Ervin S. J., Porter C. W., Bergeron R. J., Libby P. R. Differential induction of spermidine/spermine N1-acetyltransferase in human lung cancer cells by the bis(ethyl)polyamine analogues. Cancer Res. 1989 Jul 15;49(14):3829–3833. [PubMed] [Google Scholar]
  12. Casero R. A., Jr, Pegg A. E. Spermidine/spermine N1-acetyltransferase--the turning point in polyamine metabolism. FASEB J. 1993 May;7(8):653–661. [PubMed] [Google Scholar]
  13. Dai H., Kramer D. L., Yang C., Murti K. G., Porter C. W., Cleveland J. L. The polyamine oxidase inhibitor MDL-72,527 selectively induces apoptosis of transformed hematopoietic cells through lysosomotropic effects. Cancer Res. 1999 Oct 1;59(19):4944–4954. [PubMed] [Google Scholar]
  14. Gramzinski R. A., Parchment R. E., Pierce G. B. Evidence linking programmed cell death in the blastocyst to polyamine oxidation. Differentiation. 1990 Mar;43(1):59–65. doi: 10.1111/j.1432-0436.1990.tb00430.x. [DOI] [PubMed] [Google Scholar]
  15. Ha H. C., Woster P. M., Yager J. D., Casero R. A., Jr The role of polyamine catabolism in polyamine analogue-induced programmed cell death. Proc Natl Acad Sci U S A. 1997 Oct 14;94(21):11557–11562. doi: 10.1073/pnas.94.21.11557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Halline A. G., Brasitus T. A. Reversed-phase high-performance liquid chromatographic method for the measurement of polyamine oxidase activity. J Chromatogr. 1990 Nov 30;533:187–194. doi: 10.1016/s0378-4347(00)82201-x. [DOI] [PubMed] [Google Scholar]
  17. Hu R. H., Pegg A. E. Rapid induction of apoptosis by deregulated uptake of polyamine analogues. Biochem J. 1997 Nov 15;328(Pt 1):307–316. doi: 10.1042/bj3280307. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hölttä E. Oxidation of spermidine and spermine in rat liver: purification and properties of polyamine oxidase. Biochemistry. 1977 Jan 11;16(1):91–100. doi: 10.1021/bi00620a015. [DOI] [PubMed] [Google Scholar]
  19. Lee Y., Sayre L. M. Reaffirmation that metabolism of polyamines by bovine plasma amine oxidase occurs strictly at the primary amino termini. J Biol Chem. 1998 Jul 31;273(31):19490–19494. doi: 10.1074/jbc.273.31.19490. [DOI] [PubMed] [Google Scholar]
  20. Libby P. R., Porter C. W. Separation of two isozymes of polyamine oxidase from murine L1210 leukemia cells. Biochem Biophys Res Commun. 1987 Apr 14;144(1):528–535. doi: 10.1016/s0006-291x(87)80541-7. [DOI] [PubMed] [Google Scholar]
  21. Linsalata M., Cavallini A., Di Leo A. Polyamine oxidase activity and polyamine levels in human colorectal cancer and in normal surrounding mucosa. Anticancer Res. 1997 Sep-Oct;17(5B):3757–3760. [PubMed] [Google Scholar]
  22. Matsui I., Wiegand L., Pegg A. E. Properties of spermidine N-acetyltransferase from livers of rats treated with carbon tetrachloride and its role in the conversion of spermidine into putrescine. J Biol Chem. 1981 Mar 10;256(5):2454–2459. [PubMed] [Google Scholar]
  23. McCloskey D. E., Pegg A. E. Altered spermidine/spermine N1-acetyltransferase activity as a mechanism of cellular resistance to bis(ethyl)polyamine analogues. J Biol Chem. 2000 Sep 15;275(37):28708–28714. doi: 10.1074/jbc.M004120200. [DOI] [PubMed] [Google Scholar]
  24. Modrek B., Resch A., Grasso C., Lee C. Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res. 2001 Jul 1;29(13):2850–2859. doi: 10.1093/nar/29.13.2850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Müller S., Walter R. D. Purification and characterization of polyamine oxidase from Ascaris suum. Biochem J. 1992 Apr 1;283(Pt 1):75–80. doi: 10.1042/bj2830075. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Pietilä M., Alhonen L., Halmekytö M., Kanter P., Jänne J., Porter C. W. Activation of polyamine catabolism profoundly alters tissue polyamine pools and affects hair growth and female fertility in transgenic mice overexpressing spermidine/spermine N1-acetyltransferase. J Biol Chem. 1997 Jul 25;272(30):18746–18751. doi: 10.1074/jbc.272.30.18746. [DOI] [PubMed] [Google Scholar]
  27. Porter C. W., Ganis B., Libby P. R., Bergeron R. J. Correlations between polyamine analogue-induced increases in spermidine/spermine N1-acetyltransferase activity, polyamine pool depletion, and growth inhibition in human melanoma cell lines. Cancer Res. 1991 Jul 15;51(14):3715–3720. [PubMed] [Google Scholar]
  28. Quash G., Keolouangkhot T., Gazzolo L., Ripoll H., Saez S. Diamine oxidase and polyamine oxidase activities in normal and transformed cells. Biochem J. 1979 Jan 1;177(1):275–282. doi: 10.1042/bj1770275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Sarhan S., Quemener V., Moulinoux J. P., Knödgen B., Seiler N. On the degradation and elimination of spermine by the vertebrate organism. Int J Biochem. 1991;23(5-6):617–626. doi: 10.1016/0020-711x(87)90057-7. [DOI] [PubMed] [Google Scholar]
  30. Sarkar G., Sommer S. S. The "megaprimer" method of site-directed mutagenesis. Biotechniques. 1990 Apr;8(4):404–407. [PubMed] [Google Scholar]
  31. Schreuder H. A., van der Laan J. M., Swarte M. B., Kalk K. H., Hol W. G., Drenth J. Crystal structure of the reduced form of p-hydroxybenzoate hydroxylase refined at 2.3 A resolution. Proteins. 1992 Oct;14(2):178–190. doi: 10.1002/prot.340140205. [DOI] [PubMed] [Google Scholar]
  32. Seiler N., Badolo L., Duranton B., Vincent F., Schneider Y., Gossé F., Raul F. Effect of the polyamine oxidase inactivator MDL 72527 on N(1)-(n-octanesulfonyl)spermine toxicity. Int J Biochem Cell Biol. 2000 Oct;32(10):1055–1068. doi: 10.1016/s1357-2725(00)00052-2. [DOI] [PubMed] [Google Scholar]
  33. Seiler N., Bolkenius F. N., Rennert O. M. Interconversion, catabolism and elimination of the polyamines. Med Biol. 1981 Dec;59(5-6):334–346. [PubMed] [Google Scholar]
  34. Seiler N., Douaud F., Renault J., Delcros J. G., Havouis R., Uriac P., Moulinoux J. P. Polyamine sulfonamides with NMDA antagonist properties are potent calmodulin antagonists and cytotoxic agents. Int J Biochem Cell Biol. 1998 Mar;30(3):393–406. doi: 10.1016/s1357-2725(97)00150-7. [DOI] [PubMed] [Google Scholar]
  35. Seiler N. Functions of polyamine acetylation. Can J Physiol Pharmacol. 1987 Oct;65(10):2024–2035. doi: 10.1139/y87-317. [DOI] [PubMed] [Google Scholar]
  36. Seiler N. Polyamine oxidase, properties and functions. Prog Brain Res. 1995;106:333–344. doi: 10.1016/s0079-6123(08)61229-7. [DOI] [PubMed] [Google Scholar]
  37. Shappell N. W., Miller J. T., Bergeron R. J., Porter C. W. Differential effects of the spermine analog, N1, N12-bis(ethyl)-spermine, on polyamine metabolism and cell growth in human melanoma cell lines and melanocytes. Anticancer Res. 1992 Jul-Aug;12(4):1083–1089. [PubMed] [Google Scholar]
  38. TABOR C. W., TABOR H., BACHRACH U. IDENTIFICATION OF THE AMINOALDEHYDES PRODUCED BY THE OXIDATION OF SPERMINE AND SPERMIDINE WITH PURIFIED PLASMA AMINE OXIDASE. J Biol Chem. 1964 Jul;239:2194–2203. [PubMed] [Google Scholar]
  39. Takenoshita S., Matsuzaki S., Nakano G., Kimura H., Hoshi H., Shoda H., Nakamura T. Selective elevation of the N1-acetylspermidine level in human colorectal adenocarcinomas. Cancer Res. 1984 Feb;44(2):845–847. [PubMed] [Google Scholar]
  40. Tavladoraki P., Schininà M. E., Cecconi F., Di Agostino S., Manera F., Rea G., Mariottini P., Federico R., Angelini R. Maize polyamine oxidase: primary structure from protein and cDNA sequencing. FEBS Lett. 1998 Apr 10;426(1):62–66. doi: 10.1016/s0014-5793(98)00311-1. [DOI] [PubMed] [Google Scholar]
  41. Tsukada T., Furusako S., Maekawa S., Hibasami H., Nakashima K. Purification by affinity chromatography and characterization of porcine liver cytoplasmic polyamine oxidase. Int J Biochem. 1988;20(7):695–702. doi: 10.1016/0020-711x(88)90164-4. [DOI] [PubMed] [Google Scholar]
  42. Vujcic S., Halmekyto M., Diegelman P., Gan G., Kramer D. L., Janne J., Porter C. W. Effects of conditional overexpression of spermidine/spermine N1-acetyltransferase on polyamine pool dynamics, cell growth, and sensitivity to polyamine analogs. J Biol Chem. 2000 Dec 8;275(49):38319–38328. doi: 10.1074/jbc.M003270200. [DOI] [PubMed] [Google Scholar]
  43. Wallace H. M., Duthie J., Evans D. M., Lamond S., Nicoll K. M., Heys S. D. Alterations in polyamine catabolic enzymes in human breast cancer tissue. Clin Cancer Res. 2000 Sep;6(9):3657–3661. [PubMed] [Google Scholar]
  44. Wang Y., Devereux W., Woster P. M., Stewart T. M., Hacker A., Casero R. A., Jr Cloning and characterization of a human polyamine oxidase that is inducible by polyamine analogue exposure. Cancer Res. 2001 Jul 15;61(14):5370–5373. [PubMed] [Google Scholar]
  45. White W. H., Gunyuzlu P. L., Toyn J. H. Saccharomyces cerevisiae is capable of de Novo pantothenic acid biosynthesis involving a novel pathway of beta-alanine production from spermine. J Biol Chem. 2001 Jan 11;276(14):10794–10800. doi: 10.1074/jbc.M009804200. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES