Neurochemical Research

, Volume 20, Issue 10, pp 1233–1237 | Cite as

Partial purification of a phosphoethanolamine methyltransferase from rat brain cytosol

  • Subrata Mukherjee
  • Louis Freysz
  • Julian N. Kanfer
Original Articles

Abstract

The conversion of phosphoethanolamine to phosphocholine requires 3 separate N-methyltransferases. We had previously purified the enzyme catalyzing the last methylation, phosphodimethylethanolamine N-methyltransferase. We have successfully purified the enzyme catalyzing the initial methylation of phosphoethanolamine. A 434 fold purified enzyme from rat brain was obtained by the sequential use of ammonium sulfate fractionation, Q-Sepharose fast flow column chromatography and a ω-aminoethyl agarose column chromatography. The pH optimum was 11 or greater, the Km value for phosphoethanolamine was 167.8±41.7 μM and the Vmax was 487.3±85 mmoles/mg/hr. The kinetics for S-adenosyl-methionine, the methyldonor, has characteristics of cooperative binding with a Km of 1.805±0.59 mM and a Vmax of 16.9±3.6 μmoles/mg/hr. The activity was stimulated 6 fold by 2.5 mM MnCl2 and inhibited by DZA and S-adenosylhomocysteine. These results reinforce the early in vivo observations which had provided suggestive evidence for the existence of a pathway for the methylation of phosphoethanolamine to phosphocholine in rat brain.

Key words

Phosphoethanolamine methytransferase choline rat brain 

Abbreviations used

Adomet

S-adenosylmethionine

AdoHcy

S-adenosyl-homocysteine

CAPS

3-(cyclohexyl)amino-1-propanesulphonic acid

Cho

choline

3-DZA

3-deazaadenosine

Etn

ethanolamine

N-MT

N-methyltransferase

PEG

polyethyleneglycol

PMSF

phenylmethanesulphonyl fluoride

PEtn

phosphoethanolamine

PCho

phosphocholine

PMe2Etn

phosphodimethylethanolamine

PtdCho

phosphatidylcholine

PtdEtn

phosphatidylethanolamine

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References

  1. 1.
    Zeisel, S. H., and Blusztajn, J. K. 1994. Choline and Human Numition. Annu. Rev. Nutr. 14:269–296.Google Scholar
  2. 2.
    Ridgway, N. D. 1989. Phosphatidylethanolamine N-methyltransferase. Pages 103–120,in Vance, D. E., (ed.), Phosphatidyl-choline metabolism. CRC Press.Google Scholar
  3. 3.
    Andriamampandry, C., Freysz, L., Kanfer, J. N., Dreyfus, H., and Massarelli, R. 1989. Conversion of ethanolamine, monomethyleanolamine and dimethylethanolamine to choline-containing compounds by neurons in culture and by the rat brain. Biochem. J. 264:555–562.Google Scholar
  4. 4.
    Datko, A. H., and Mudd, S. H., 1988. Enzymes of phosphatidylcholine synthesis in lemna, soybean and carrot. Plant Physiol. 88:854–861.Google Scholar
  5. 5.
    Datko, A. H., Aksamet, R. R., and Mudd, S. H., 1990. Phosphatidyl-choline synthesis in the rat: The substrate for methylation and regulation by choline. Lipids. 25:135–142.Google Scholar
  6. 6.
    Andriamampandry, C., Massarelli, R., Freysz, L., and Kanfer, J. N., 1990. A rat brain cytosolic N-methyltransferase(s) activity converting phosphorylethanolamine into phosphorylcholine. Biochem. Biophys. Res. Commun. 171:758–763.Google Scholar
  7. 7.
    Andriamampandry, C., Massarelli, R., and Kanfer, J. N. 1992. Properties of a partially purified phosphodimethylethanolamine methyltransferase from rat brain cytosol. Biochem. J. 288:267–272.Google Scholar
  8. 8.
    Lowry, O. H., Rosenbrough, N. J., Farr, A. L., and Randall, R. J., 1951. Protein measurement with the folin phenol reagent. J. Biol. Chem. 193:265–275.Google Scholar
  9. 9.
    Sedmak, J. J., and Grossberg, S. E., 1977. A rapid, Sensitive and versatile assay for protein using Coomassie Brilliant blue G250. Anal. Biochem. 79:544–552.Google Scholar
  10. 10.
    Lineweaver, H., and Burke, D., 1934. Determination of enzyme dissociation constants. J. Amer. Chem. Soc. 56:658–666.Google Scholar
  11. 11.
    Laemmli, U. K., 1970. Cleavage of structural proteins during the assembly of head bacteriophage T4. Nature 277:680–685.Google Scholar
  12. 12.
    Kent, C., 1990. Regulation of phospholipid metabolism. Prog. Lipid Res. 29:87–105.Google Scholar
  13. 13.
    Kanfer, J. N., 1989. Phospholipase D and the base exchange enzyme in phosphatidylcholine metabolism. (Vance, D. E. ed.), pp. 65–86, CRC Press.Google Scholar
  14. 14.
    Segel, I. H. 1975. Enzyme Kinetics-Behavior and Analysis of rapid equilibrium and steady steady state enzyme systems. Pages 356–366, John Wiley and Sons, New York.Google Scholar

Copyright information

© Plenum Publishing Corporation 1995

Authors and Affiliations

  • Subrata Mukherjee
    • 1
  • Louis Freysz
    • 2
  • Julian N. Kanfer
    • 1
  1. 1.Departmet of Biochemistry and Molecular BiologyUniversity of ManitobaWinnipegCanada
  2. 2.Cenre d'NeurochimieStrasbourgFrance

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