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Expression and Functional Interaction of the Catalytic and Regulatory Subunits of Human Methionine Adenosyltransferase in Mammalian Cells

Halim, Abdel-Baset ; LeGros, Leighton ; Geller, Arthur ; Kotb, Malak

The Journal of biological chemistry, 1999-10, Vol.274 (42), p.29720-29725 [Periódico revisado por pares]

United States: Elsevier Inc

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  • Título:
    Expression and Functional Interaction of the Catalytic and Regulatory Subunits of Human Methionine Adenosyltransferase in Mammalian Cells
  • Autor: Halim, Abdel-Baset ; LeGros, Leighton ; Geller, Arthur ; Kotb, Malak
  • Assuntos: Animals ; Catalytic Domain ; Chlorocebus aethiops ; COS Cells ; Humans ; Kinetics ; Methionine Adenosyltransferase - metabolism ; Recombinant Proteins - metabolism
  • É parte de: The Journal of biological chemistry, 1999-10, Vol.274 (42), p.29720-29725
  • Notas: ObjectType-Article-2
    SourceType-Scholarly Journals-1
    ObjectType-Feature-1
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    ObjectType-Article-1
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  • Descrição: Methionine adenosyltransferase (MAT) catalyzes the synthesis of S-adenosylmethionine (AdoMet). The mammalian MAT II isozyme consists of catalytic α2 and regulatory β subunits. The aim of this study was to investigate the interaction and kinetic behavior of the human MAT II subunit proteins in mammalian cells. COS-1 cells were transiently transfected with pTargeT vector harboring full-length cDNA that encodes for the MAT II α2 or β subunits. Expression of the His-tagged recombinant α2 (rα2) subunit in COS-1 cells markedly increased MAT II activity and resulted in a shift in theKm for l-methionine (l-Met) from 15 μm (endogenous MAT II) to 75 μm(rα2), and with the apparent existence of two kinetic forms of MAT in the transfected COS-1 cell extracts. By contrast, expression of the recombinant β (rβ) subunit had no effect on theKm for l-Met of the endogenous MAT II, while it did cause an increase in both the Vmaxand the specific activity of endogenous MAT. Co-expression of both rα2 and rβ subunits resulted in a significant increase of MAT specific activity with the appearance of a single kinetic form of MAT (Km = 20 μm). The recombinant MAT II α2 and rβ subunit associated spontaneously either in cell-free system or in COS-1 cells co-expressing both subunits. Analysis of nickel-agarose-purified His-tagged rα2 subunit from COS-1 cell extracts showed that the β subunit co-purified with the α2 subunit. Furthermore, the α2 and β subunits co-migrated in native polyacrylamide gels. Together, the data provide evidence for α2 and β MAT subunit association. In addition, the β subunit regulated MAT II activity by reducing its Km for l-Met and by rendering the enzyme more susceptible to feedback inhibition by AdoMet. We believe that the previously described differential expression of MAT II β subunit may be an important mechanism by which MAT activity can be modulated to provide different levels of AdoMet that may be required at different stages of cell growth and differentiation.
  • Editor: United States: Elsevier Inc
  • Idioma: Inglês
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  • Realização: ABCD-USP USP   Logos de Redes Sociais: Freepik

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