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. 1996 Oct 1;319(Pt 1):21–26. doi: 10.1042/bj3190021

Substrate protection against inactivation of the mammalian polyamine-transport system by 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide.

K Torossian 1, M Audette 1, R Poulin 1
PMCID: PMC1217730  PMID: 8870644

Abstract

Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11 +/- 4 and 96 +/- 16 microM after 30 min at 22 degrees C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and K(m) for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 degrees C respectively. Spermine (10 microM) almost fully protected against spermidine-transport inactivation by EDC at 22 degrees C, and decreased the rate of inactivation at 37 degrees C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 degrees C with EC50 values estimated at 10, 1 and less than 1 microM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89 +/- 3 compared with 44 +/- 5%), whereas the converse was true for 100 microM DCC (81 +/- 3 compared with 92 +/- 5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na(+)-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to crosslink a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.

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Selected References

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  1. Bass R., Hedegaard H. B., Dillehay L., Moffett J., Englesberg E. The A, ASC, and L systems for the transport of amino acids in Chinese hamster ovary cells (CHO-K1). J Biol Chem. 1981 Oct 25;256(20):10259–10266. [PubMed] [Google Scholar]
  2. Bauminger S., Wilchek M. The use of carbodiimides in the preparation of immunizing conjugates. Methods Enzymol. 1980;70(A):151–159. doi: 10.1016/s0076-6879(80)70046-0. [DOI] [PubMed] [Google Scholar]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Byers T. L., Pegg A. E. Properties and physiological function of the polyamine transport system. Am J Physiol. 1989 Sep;257(3 Pt 1):C545–C553. doi: 10.1152/ajpcell.1989.257.3.C545. [DOI] [PubMed] [Google Scholar]
  5. Byers T. L., Pegg A. E. Regulation of polyamine transport in Chinese hamster ovary cells. J Cell Physiol. 1990 Jun;143(3):460–467. doi: 10.1002/jcp.1041430309. [DOI] [PubMed] [Google Scholar]
  6. Chang B. K., Libby P. R., Bergeron R. J., Porter C. W. Modulation of polyamine biosynthesis and transport by oncogene transfection. Biochem Biophys Res Commun. 1988 Nov 30;157(1):264–270. doi: 10.1016/s0006-291x(88)80042-1. [DOI] [PubMed] [Google Scholar]
  7. Craik J. D., Reithmeier R. A. Reversible and irreversible inhibition of phosphate transport in human erythrocytes by a membrane impermeant carbodiimide. J Biol Chem. 1985 Feb 25;260(4):2404–2408. [PubMed] [Google Scholar]
  8. Forgac M. Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev. 1989 Jul;69(3):765–796. doi: 10.1152/physrev.1989.69.3.765. [DOI] [PubMed] [Google Scholar]
  9. Furuchi T., Kashiwagi K., Kobayashi H., Igarashi K. Characteristics of the gene for a spermidine and putrescine transport system that maps at 15 min on the Escherichia coli chromosome. J Biol Chem. 1991 Nov 5;266(31):20928–20933. [PubMed] [Google Scholar]
  10. Grabarek Z., Gergely J. Zero-length crosslinking procedure with the use of active esters. Anal Biochem. 1990 Feb 15;185(1):131–135. doi: 10.1016/0003-2697(90)90267-d. [DOI] [PubMed] [Google Scholar]
  11. Honig B., Nicholls A. Classical electrostatics in biology and chemistry. Science. 1995 May 26;268(5214):1144–1149. doi: 10.1126/science.7761829. [DOI] [PubMed] [Google Scholar]
  12. Johnson M. D., Ash J. F., Pauw P. G. Alterations in amino acid transport in Na,K-ATPase amplified HeLa cells. J Biol Chem. 1986 Jan 5;261(1):40–43. [PubMed] [Google Scholar]
  13. Kakinuma Y., Hoshino K., Igarashi K. Characterization of the inducible polyamine transporter in bovine lymphocytes. Eur J Biochem. 1988 Sep 15;176(2):409–414. doi: 10.1111/j.1432-1033.1988.tb14297.x. [DOI] [PubMed] [Google Scholar]
  14. Kim Y. K., Kim T. I., Jung D. K., Jung J. S., Lee S. H. Inhibition of H+/organic cation antiport by carboxyl reagents in rabbit renal brush-border membrane vesicles. J Pharmacol Exp Ther. 1993 Aug;266(2):500–505. [PubMed] [Google Scholar]
  15. Kumagai J., Jain R., Johnson L. R. Characteristics of spermidine uptake by isolated rat enterocytes. Am J Physiol. 1989 May;256(5 Pt 1):G905–G910. doi: 10.1152/ajpgi.1989.256.5.G905. [DOI] [PubMed] [Google Scholar]
  16. Lessard M., Zhao C., Singh S. M., Poulin R. Hormonal and feedback regulation of putrescine and spermidine transport in human breast cancer cells. J Biol Chem. 1995 Jan 27;270(4):1685–1694. [PubMed] [Google Scholar]
  17. Marton L. J., Pegg A. E. Polyamines as targets for therapeutic intervention. Annu Rev Pharmacol Toxicol. 1995;35:55–91. doi: 10.1146/annurev.pa.35.040195.000415. [DOI] [PubMed] [Google Scholar]
  18. Moffett J. R., Namboodiri M. A., Neale J. H. Enhanced carbodiimide fixation for immunohistochemistry: application to the comparative distributions of N-acetylaspartylglutamate and N-acetylaspartate immunoreactivities in rat brain. J Histochem Cytochem. 1993 Apr;41(4):559–570. doi: 10.1177/41.4.8450195. [DOI] [PubMed] [Google Scholar]
  19. Ohsumi Y., Anraku Y. Calcium transport driven by a proton motive force in vacuolar membrane vesicles of Saccharomyces cerevisiae. J Biol Chem. 1983 May 10;258(9):5614–5617. [PubMed] [Google Scholar]
  20. Osborne D. L., Seidel E. R. Gastrointestinal luminal polyamines: cellular accumulation and enterohepatic circulation. Am J Physiol. 1990 Apr;258(4 Pt 1):G576–G584. doi: 10.1152/ajpgi.1990.258.4.G576. [DOI] [PubMed] [Google Scholar]
  21. Ovchinnikov YuA, Abdulaev N. G., Modyanov N. N. Structural basis of proton-translocating protein function. Annu Rev Biophys Bioeng. 1982;11:445–463. doi: 10.1146/annurev.bb.11.060182.002305. [DOI] [PubMed] [Google Scholar]
  22. Pedemonte C. H., Kaplan J. H. Carbodiimide inactivation of Na,K-ATPase. A consequence of internal cross-linking and not carboxyl group modification. J Biol Chem. 1986 Mar 15;261(8):3632–3639. [PubMed] [Google Scholar]
  23. Pedemonte C. H., Kaplan J. H. Chemical modification as an approach to elucidation of sodium pump structure-function relations. Am J Physiol. 1990 Jan;258(1 Pt 1):C1–23. doi: 10.1152/ajpcell.1990.258.1.C1. [DOI] [PubMed] [Google Scholar]
  24. Persson L., Holm I., Ask A., Heby O. Curative effect of DL-2-difluoromethylornithine on mice bearing mutant L1210 leukemia cells deficient in polyamine uptake. Cancer Res. 1988 Sep 1;48(17):4807–4811. [PubMed] [Google Scholar]
  25. Pistocchi R., Kashiwagi K., Miyamoto S., Nukui E., Sadakata Y., Kobayashi H., Igarashi K. Characteristics of the operon for a putrescine transport system that maps at 19 minutes on the Escherichia coli chromosome. J Biol Chem. 1993 Jan 5;268(1):146–152. [PubMed] [Google Scholar]
  26. Poulin R., Lessard M., Zhao C. Inorganic cation dependence of putrescine and spermidine transport in human breast cancer cells. J Biol Chem. 1995 Jan 27;270(4):1695–1704. doi: 10.1074/jbc.270.4.1695. [DOI] [PubMed] [Google Scholar]
  27. Rinehart C. A., Jr, Chen K. Y. Characterization of the polyamine transport system in mouse neuroblastoma cells. Effects of sodium and system A amino acids. J Biol Chem. 1984 Apr 25;259(8):4750–4756. [PubMed] [Google Scholar]
  28. Seiler N., Dezeure F. Polyamine transport in mammalian cells. Int J Biochem. 1990;22(3):211–218. doi: 10.1016/0020-711x(90)90332-w. [DOI] [PubMed] [Google Scholar]
  29. Seiler N., Sarhan S., Grauffel C., Jones R., Knödgen B., Moulinoux J. P. Endogenous and exogenous polyamines in support of tumor growth. Cancer Res. 1990 Aug 15;50(16):5077–5083. [PubMed] [Google Scholar]
  30. Suchi R., Stern-Bach Y., Gabay T., Schuldiner S. Covalent modification of the amine transporter with N,N'-dicyclohexylcarbodiimide. Biochemistry. 1991 Jul 2;30(26):6490–6494. doi: 10.1021/bi00240a020. [DOI] [PubMed] [Google Scholar]
  31. Sussman M. R., Slayman C. W. Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexylcarbodiimide. J Biol Chem. 1983 Feb 10;258(3):1839–1843. [PubMed] [Google Scholar]

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