RESUMO
Myxococcus xanthus Nudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8-oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn2+ was the most effective co-factor for stimulating oxidized deoxynucleotide hydrolase activity. The Km of Nud2 with 8-oxo-dGTP for Mn2+ was 19-fold lower than that for Mg2+, and was 2-fold lower than that with dGTP for Mn2+. The specificity constant (kcat/Km) for 8-oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif (84AX590GX7REX2EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.
Assuntos
Myxococcus xanthus/enzimologia , Pirofosfatases/metabolismo , Proteínas de Bactérias/isolamento & purificação , Proteínas de Bactérias/metabolismo , Meios de Cultura/química , Enzimas Reparadoras do DNA/metabolismo , Nucleotídeos de Desoxiguanina/metabolismo , Hidrólise , Cinética , Mutação , Myxococcus xanthus/genética , Oxirredução , Monoéster Fosfórico Hidrolases/metabolismo , Pirofosfatases/isolamento & purificação , Especificidade por Substrato , Nudix HidrolasesAssuntos
Proteínas de Bactérias/metabolismo , Myxococcus xanthus/enzimologia , Pirofosfatases/metabolismo , Proteínas de Bactérias/genética , Cátions Bivalentes , Fosfatos de Dinucleosídeos/metabolismo , Hidrólise , Myxococcus xanthus/genética , Pirofosfatases/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Nudix HidrolasesRESUMO
PKCdelta was revealed to make a homologous protein complex that shows a high protein kinase activity upon H(2)O(2) stimulation by expressing the enzymes having different epitope tags in COS-7 cells. The association of the endogenous PKCdelta in the cells was observed by sucrose density gradients. Analysis using the mutant replacing the tyrosine phosphorylation sites showed that PKCdelta is activated without tyrosine phosphorylation in the stimulated cells, and the time course of the activation was parallel with that of the complex formation. The binding sites were identified as the C1 and C2-like regions in the regulatory domain using a series of deletion mutants. The binding between the C1 and C2-like region fragments was induced by cell stimulation, whereas the association of the C1 region fragments by itself and that of the C2-like region fragments were observed even without stimulation. These results suggest that the protein complexes of PKCdelta through the association between the C1 and C2-like regions by different combinations are generated in the H(2)O(2)-treated cells, that may show an enhanced protein kinase activity.