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. 1999 Jul 1;341(Pt 1):133–138.

Co-ordinate variations in methylmalonyl-CoA mutase and methionine synthase, and the cobalamin cofactors in human glioma cells during nitrous oxide exposure and the subsequent recovery phase.

B Riedel 1, T Fiskerstrand 1, H Refsum 1, P M Ueland 1
PMCID: PMC1220339  PMID: 10377254

Abstract

We investigated the co-ordinate variations of the two cobalamin (Cbl)-dependent enzymes, methionine synthase (MS) and methylmalonyl-CoA mutase (MCM), and measured the levels of their respective cofactors, methylcobalamin (CH3Cbl) and adenosylcobalamin (AdoCbl) in cultured human glioma cells during nitrous oxide exposure and during a subsequent recovery period of culture in a nitrous oxide-free atmosphere (air). In agreement with published data, MS as the primary target of nitrous oxide was inactivated rapidly (initial rate of 0.06 h(-1)), followed by reduction of CH3Cbl (to <20%). Both enzyme activity and cofactor levels recovered rapidly when the cells were subsequently cultured in air, but the recovery was completely blocked by the protein-synthesis inhibitor, cycloheximide. During MS inactivation, there was a reduction of cellular AdoCbl and holo-MCM activity (measured in the absence of exogenous AdoCbl) to about 50% of pre-treatment levels. When the cells were transferred to air, both AdoCbl and holo-MCM activity recovered, albeit more slowly than the MS system. Notably, the regain of the holo-MCM and AdoCbl was enhanced rather than inhibited by cycloheximide. These findings confirm irreversible damage of MS by nitrous oxide; hence, synthesis of the enzyme is required to restore its activity. In contrast, restoration of holo-MCM activity is only dependent on repletion of the AdoCbl cofactor. We also observed a synchronous fluctuation in AdoCbl and the much larger hydroxycobalamin pool during the inactivation and recovery phase, suggesting that the loss and repletion of AdoCbl reflect changes in intracellular Cbl homoeostasis. Our data demonstrate that the nitrous oxide-induced changes in MS and CH3Cbl are associated with reversible changes in both MCM holoactivity and the AdoCbl level, suggesting co-ordinate distribution of Cbl cofactors during depletion and repletion.

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Selected References

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  1. Akslen L. A., Andersen K. J., Bjerkvig R. Characteristics of human and rat glioma cells grown in a defined medium. Anticancer Res. 1988 Jul-Aug;8(4):797–803. [PubMed] [Google Scholar]
  2. Banerjee R. V., Matthews R. G. Cobalamin-dependent methionine synthase. FASEB J. 1990 Mar;4(5):1450–1459. doi: 10.1096/fasebj.4.5.2407589. [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. Cardinale G. J., Dreyfus P. M., Auld P., Abeles R. H. Experimental vitamin B12 deficiency: its effect on tissue vitamin B12-coenzyme levels and on the metabolism of methylmalonyl-CoA. Arch Biochem Biophys. 1969 Apr;131(1):92–99. doi: 10.1016/0003-9861(69)90108-8. [DOI] [PubMed] [Google Scholar]
  5. Christensen B., Guttormsen A. B., Schneede J., Riedel B., Refsum H., Svardal A., Ueland P. M. Preoperative methionine loading enhances restoration of the cobalamin-dependent enzyme methionine synthase after nitrous oxide anesthesia. Anesthesiology. 1994 May;80(5):1046–1056. doi: 10.1097/00000542-199405000-00014. [DOI] [PubMed] [Google Scholar]
  6. Christensen B., Refsum H., Garras A., Ueland P. M. Homocysteine remethylation during nitrous oxide exposure of cells cultured in media containing various concentrations of folates. J Pharmacol Exp Ther. 1992 Jun;261(3):1096–1105. [PubMed] [Google Scholar]
  7. Christensen B., Refsum H., Vintermyr O., Ueland P. M. Homocysteine export from cells cultured in the presence of physiological or superfluous levels of methionine: methionine loading of non-transformed, transformed, proliferating, and quiescent cells in culture. J Cell Physiol. 1991 Jan;146(1):52–62. doi: 10.1002/jcp.1041460108. [DOI] [PubMed] [Google Scholar]
  8. Christensen B., Rosenblatt D. S., Chu R. C., Ueland P. M. Effect of methionine and nitrous oxide on homocysteine export and remethylation in fibroblasts from cystathionine synthase-deficient, cb1G, and cb1E patients. Pediatr Res. 1994 Jan;35(1):3–9. doi: 10.1203/00006450-199401000-00002. [DOI] [PubMed] [Google Scholar]
  9. Christensen B., Ueland P. M. Methionine synthase inactivation by nitrous oxide during methionine loading of normal human fibroblasts. Homocysteine remethylation as determinant of enzyme inactivation and homocysteine export. J Pharmacol Exp Ther. 1993 Dec;267(3):1298–1303. [PubMed] [Google Scholar]
  10. Deacon R., Lumb M., Perry J., Chanarin I., Minty B., Halsey M., Nunn J. Inactivation of methionine synthase by nitrous oxide. Eur J Biochem. 1980 Mar;104(2):419–423. doi: 10.1111/j.1432-1033.1980.tb04443.x. [DOI] [PubMed] [Google Scholar]
  11. Drummond J. T., Matthews R. G. Nitrous oxide degradation by cobalamin-dependent methionine synthase: characterization of the reactants and products in the inactivation reaction. Biochemistry. 1994 Mar 29;33(12):3732–3741. doi: 10.1021/bi00178a033. [DOI] [PubMed] [Google Scholar]
  12. Drummond J. T., Matthews R. G. Nitrous oxide inactivation of cobalamin-dependent methionine synthase from Escherichia coli: characterization of the damage to the enzyme and prosthetic group. Biochemistry. 1994 Mar 29;33(12):3742–3750. doi: 10.1021/bi00178a034. [DOI] [PubMed] [Google Scholar]
  13. Ermens A. A., Refsum H., Rupreht J., Spijkers L. J., Guttormsen A. B., Lindemans J., Ueland P. M., Abels J. Monitoring cobalamin inactivation during nitrous oxide anesthesia by determination of homocysteine and folate in plasma and urine. Clin Pharmacol Ther. 1991 Apr;49(4):385–393. doi: 10.1038/clpt.1991.45. [DOI] [PubMed] [Google Scholar]
  14. Fiskerstrand T., Christensen B., Tysnes O. B., Ueland P. M., Refsum H. Development and reversion of methionine dependence in a human glioma cell line: relation to homocysteine remethylation and cobalamin status. Cancer Res. 1994 Sep 15;54(18):4899–4906. [PubMed] [Google Scholar]
  15. Fiskerstrand T., Riedel B., Ueland P. M., Seetharam B., Pezacka E. H., Gulati S., Bose S., Banerjee R., Berge R. K., Refsum H. Disruption of a regulatory system involving cobalamin distribution and function in a methionine-dependent human glioma cell line. J Biol Chem. 1998 Aug 7;273(32):20180–20184. doi: 10.1074/jbc.273.32.20180. [DOI] [PubMed] [Google Scholar]
  16. Fiskerstrand T., Ueland P. M., Refsum H. Folate depletion induced by methotrexate affects methionine synthase activity and its susceptibility to inactivation by nitrous oxide. J Pharmacol Exp Ther. 1997 Sep;282(3):1305–1311. [PubMed] [Google Scholar]
  17. Frasca V., Riazzi B. S., Matthews R. G. In vitro inactivation of methionine synthase by nitrous oxide. J Biol Chem. 1986 Dec 5;261(34):15823–15826. [PubMed] [Google Scholar]
  18. Guttormsen A. B., Refsum H., Ueland P. M. The interaction between nitrous oxide and cobalamin. Biochemical effects and clinical consequences. Acta Anaesthesiol Scand. 1994 Nov;38(8):753–756. doi: 10.1111/j.1399-6576.1994.tb03996.x. [DOI] [PubMed] [Google Scholar]
  19. Kano Y., Sakamoto S., Sakuraya K., Kubota T., Hida K., Suda K., Takaku F. Effect of nitrous oxide on human bone marrow cells and its synergistic effect with methionine and methotrexate on functional folate deficiency. Cancer Res. 1981 Nov;41(11 Pt 1):4698–4701. [PubMed] [Google Scholar]
  20. Kennedy D. G., Cannavan A., Molloy A., O'Harte F., Taylor S. M., Kennedy S., Blanchflower W. J. Methylmalonyl-CoA mutase (EC 5.4.99.2) and methionine synthetase (EC 2.1.1.13) in the tissues of cobalt-vitamin B12 deficient sheep. Br J Nutr. 1990 Nov;64(3):721–732. doi: 10.1079/bjn19900074. [DOI] [PubMed] [Google Scholar]
  21. Kennedy D. G., Young P. B., Kennedy S., Scott J. M., Molloy A. M., Weir D. G., Price J. Cobalt-vitamin B12 deficiency and the activity of methylmalonyl CoA mutase and methionine synthase in cattle. Int J Vitam Nutr Res. 1995;65(4):241–247. [PubMed] [Google Scholar]
  22. Koblin D. D., Waskell L., Watson J. E., Stokstad E. L., Eger E. I., 2nd Nitrous oxide inactivates methionine synthetase in human liver. Anesth Analg. 1982 Feb;61(2):75–78. [PubMed] [Google Scholar]
  23. Koblin D. D., Watson J. E., Deady J. E., Stokstad E. L., Eger E. I., 2nd Inactivation of methionine synthetase by nitrous oxide in mice. Anesthesiology. 1981 Apr;54(4):318–324. doi: 10.1097/00000542-198104000-00012. [DOI] [PubMed] [Google Scholar]
  24. Kondo H., Osborne M. L., Kolhouse J. F., Binder M. J., Podell E. R., Utley C. S., Abrams R. S., Allen R. H. Nitrous oxide has multiple deleterious effects on cobalamin metabolism and causes decreases in activities of both mammalian cobalamin-dependent enzymes in rats. J Clin Invest. 1981 May;67(5):1270–1283. doi: 10.1172/JCI110155. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Landon M. J., Toothill V. J. Effect of nitrous oxide on placental methionine synthase activity. Br J Anaesth. 1986 May;58(5):524–527. doi: 10.1093/bja/58.5.524. [DOI] [PubMed] [Google Scholar]
  26. Ludwig M. L., Matthews R. G. Structure-based perspectives on B12-dependent enzymes. Annu Rev Biochem. 1997;66:269–313. doi: 10.1146/annurev.biochem.66.1.269. [DOI] [PubMed] [Google Scholar]
  27. Molloy A. M., Orsi B., Kennedy D. G., Kennedy S., Weir D. G., Scott J. M. The relationship between the activity of methionine synthase and the ratio of S-adenosylmethionine to S-adenosylhomocysteine in the brain and other tissues of the pig. Biochem Pharmacol. 1992 Oct 6;44(7):1349–1355. doi: 10.1016/0006-2952(92)90536-r. [DOI] [PubMed] [Google Scholar]
  28. Parry T. E., Laurence A. S., Blackmore J. A., Roberts B. Serum valine, methionine and isoleucine levels in patients anaesthetized with and without nitrous oxide. Clin Lab Haematol. 1985;7(4):317–326. doi: 10.1111/j.1365-2257.1985.tb00046.x. [DOI] [PubMed] [Google Scholar]
  29. Rask H., Olesen A. S., Mortensen J. Z., Freund L. G. N2O and urine methylmalonic acid in man. Scand J Haematol. 1983 Jul;31(1):45–48. doi: 10.1111/j.1600-0609.1983.tb02135.x. [DOI] [PubMed] [Google Scholar]
  30. Riedel B., Ueland P. M., Svardal A. M. Fully automated assay for cobalamin-dependent methylmalonyl CoA mutase. Clin Chem. 1995 Aug;41(8 Pt 1):1164–1170. [PubMed] [Google Scholar]
  31. Royston B. D., Nunn J. F., Weinbren H. K., Royston D., Cormack R. S. Rate of inactivation of human and rodent hepatic methionine synthase by nitrous oxide. Anesthesiology. 1988 Feb;68(2):213–216. doi: 10.1097/00000542-198802000-00006. [DOI] [PubMed] [Google Scholar]
  32. Stabler S. P., Brass E. P., Marcell P. D., Allen R. H. Inhibition of cobalamin-dependent enzymes by cobalamin analogues in rats. J Clin Invest. 1991 Apr;87(4):1422–1430. doi: 10.1172/JCI115148. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vineis P. Molecular epidemiology: low-dose carcinogens and genetic susceptibility. Int J Cancer. 1997 Mar 28;71(1):1–3. doi: 10.1002/(sici)1097-0215(19970328)71:1<1::aid-ijc1>3.0.co;2-y. [DOI] [PubMed] [Google Scholar]
  34. Vitols E., Walker G. A., Huennekens F. M. Enzymatic conversion of vitamin B-12s to a cobamide coenzyme, alpha-(5,6-dimethylbenzimidazolyl)deoxyadenosylcobamide (adenosyl-B-12). J Biol Chem. 1966 Apr 10;241(7):1455–1461. [PubMed] [Google Scholar]
  35. WEISSBACH H., PETERKOFSKY A., REDFIELD B. G., DICKERMAN H. STUDIES ON THE TERMINAL REACTION IN THE BIOSYNTHESIS OF METHIONINE. J Biol Chem. 1963 Oct;238:3318–3324. [PubMed] [Google Scholar]
  36. Walker G. A., Murphy S., Huennekens F. M. Enzymatic conversion of vitamin B 12a to adenosyl-B 12: evidence for the existence of two separate reducing systems. Arch Biochem Biophys. 1969 Oct;134(1):95–102. doi: 10.1016/0003-9861(69)90255-0. [DOI] [PubMed] [Google Scholar]
  37. Watanabe F., Nakano Y., Tachikake N., Saido H., Tamura Y., Yamanaka H. Vitamin B-12 deficiency increases the specific activities of rat liver NADH- and NADPH-linked aquacobalamin reductase isozymes involved in coenzyme synthesis. J Nutr. 1991 Dec;121(12):1948–1954. doi: 10.1093/jn/121.12.1948. [DOI] [PubMed] [Google Scholar]
  38. van Kapel J., Spijkers L. J., Lindemans J., Abels J. Improved distribution analysis of cobalamins and cobalamin analogues in human plasma in which the use of thiol-blocking agents is a prerequisite. Clin Chim Acta. 1983 Jul 15;131(3):211–224. doi: 10.1016/0009-8981(83)90090-6. [DOI] [PubMed] [Google Scholar]

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