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. 2012 Jun 21;40(17):8568–8578. doi: 10.1093/nar/gks593

Figure 4.

Figure 4.

GkaRloC’s ATPase activates its ACNase. GkaRloC’s ACNase of the IMAC fraction was assayed in vitro in panels (A)–(C) and (E) essentially as described in Materials and Methods but in the absence of added DNA. (A) Dependence of GkaRloC’s ACNase activity on ATP’s level. (B) GkaRloC’s ACNase activity was assayed in the presence of 500 µM of the indicated nucleotides. (C) Time courses of GkaRloC’s ACNase activity in the presence of 0.5 mM ATP and indicated amounts of AMPPNP. (D) In vivo ACNase activity of the indicated GkaRloC alleles. Left panel—RNA extracted from cells expressing these alleles was 5′-end labelled using T4 Pnk and separated by denaturing PAGE. Right panel—the expression of the indicated GkaRloC alleles were monitored by Western using an anti-His tag monoclonal antibody (4). (E) Nucleotide specificity of GkaRloC’s ACNase activation. The activation reaction was performed in the presence of the indicated nucleotides (GTP and ATP at 0.5 mM each, dTTP at 5 µM).