Purification and properties of S-adenosyl-L-methionine

L-methionine S-methyltransferase from Wollastonia biflora leaves

Franck James, Kurt D Nolte, Andrew D. Hanson

Research output: Contribution to journalArticle

48 Citations (Scopus)

Abstract

The plant enzyme S-adenosylmethionine:methionine S-methyltransferase (EC 2.1.1.12, MMT) catalyzes the synthesis of S-methylmethionine. MMT was purified 620-fold to apparent homogeneity from leaves of Wollastonia biflora. The four-step purification included fractionation with polyethylene glycol, affinity chromatography on adenosine-agarose, anion exchange chromatography, and gel filtration. Protein yield was about 180 μg/kg of leaves. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native gel filtration chromatography were, respectively, 115 and 450 kDa, suggesting a tetramer of 115-kDa subunits. The 115-kDa subunit was photoaffinity labeled by S-adenosyl[3H]methionine. Antibodies raised against W. biflora MMT recognized a 115-kDa polypeptide in partially purified MMT preparations from leaves of lettuce, cabbage, clover, and maize. The pH optimum of W. biflora MMT was 7.2. Kinetic analysis of substrate interaction and product inhibition patterns indicated an Ordered Bi Bi mechanism, with S-adenosylmethionine the first reactant to bind and S-adenosylhomocysteine the last product to be released. The enzyme catalyzed methylation of selenomethionine and ethionine, but not of S-methylcysteine, homocysteine, cysteine, or peptidylmethionine. Tests with other substrate analogs indicated that a free carboxyl group was required for enzyme activity, and that a free amino group was not.

Original languageEnglish (US)
Pages (from-to)22344-22350
Number of pages7
JournalJournal of Biological Chemistry
Volume270
Issue number38
StatePublished - Sep 22 1995
Externally publishedYes

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methionine S-methyltransferase
Purification
S-Adenosylmethionine
Chromatography
Gel Chromatography
Enzymes
Gels
S-Adenosylhomocysteine
Vitamin U
Ethionine
Selenomethionine
Native Polyacrylamide Gel Electrophoresis
Affinity chromatography
Medicago
Lettuce
Methylation
Brassica
Enzyme activity
Molecular mass
Homocysteine

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and properties of S-adenosyl-L-methionine : L-methionine S-methyltransferase from Wollastonia biflora leaves. / James, Franck; Nolte, Kurt D; Hanson, Andrew D.

In: Journal of Biological Chemistry, Vol. 270, No. 38, 22.09.1995, p. 22344-22350.

Research output: Contribution to journalArticle

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abstract = "The plant enzyme S-adenosylmethionine:methionine S-methyltransferase (EC 2.1.1.12, MMT) catalyzes the synthesis of S-methylmethionine. MMT was purified 620-fold to apparent homogeneity from leaves of Wollastonia biflora. The four-step purification included fractionation with polyethylene glycol, affinity chromatography on adenosine-agarose, anion exchange chromatography, and gel filtration. Protein yield was about 180 μg/kg of leaves. Estimates of molecular mass from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and native gel filtration chromatography were, respectively, 115 and 450 kDa, suggesting a tetramer of 115-kDa subunits. The 115-kDa subunit was photoaffinity labeled by S-adenosyl[3H]methionine. Antibodies raised against W. biflora MMT recognized a 115-kDa polypeptide in partially purified MMT preparations from leaves of lettuce, cabbage, clover, and maize. The pH optimum of W. biflora MMT was 7.2. Kinetic analysis of substrate interaction and product inhibition patterns indicated an Ordered Bi Bi mechanism, with S-adenosylmethionine the first reactant to bind and S-adenosylhomocysteine the last product to be released. The enzyme catalyzed methylation of selenomethionine and ethionine, but not of S-methylcysteine, homocysteine, cysteine, or peptidylmethionine. Tests with other substrate analogs indicated that a free carboxyl group was required for enzyme activity, and that a free amino group was not.",
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