Nickel-charged UreG dimer can activate urease <i>in vitro</i> in the presence of UreF/UreH.

<p>(A) <i>In vitro</i> urease assay was performed by incubating apo-urease with purified samples of UreF/UreH, bicarbonate ion and nickel-charged UreG dimers containing either GTP or GTPγS. In the presence of UreF/UreH, the urease was activated, with a specific activity of 605±14 nmol NH₃ mg urease⁻¹ min⁻¹, by the nickel-charged UreG dimer containing GTP, but not by the UreG dimer containing GTPγS. For the purpose of comparison, <i>H. pylori</i> urease activity has been reported to be in the range of ∼10–1,000 µmol NH₃ mg⁻¹ min⁻¹<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001678#pbio.1001678-Shi1" target="_blank">[23]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001678#pbio.1001678-Benoit1" target="_blank">[37]</a>,<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001678#pbio.1001678-Ha1" target="_blank">[58]</a>. (B) <i>In vitro</i> urease assay was performed by incubating apo-urease, UreF/UreH, and nickel-charged UreG dimers in the presence or absence of 100 mM bicarbonate. (C) <i>In vitro</i> urease assay was performed by incubating apo-urease and UreF/UreH with bicarbonate ion and 20 µM of nickel-charged UreG dimers or 20 µM free nickel. (D) GST-UreF/UreH or GST-UreF/UreH/urease was first immobilized on a 5 ml GSTrap column. Purified GTP/nickel-charged UreG dimer was loaded onto the column and incubated for 15 min. After washing to remove excess UreG, protein was eluted with glutathione and analyzed using SDS-PAGE. Nickel-charged UreG dimer does not interact with the GST-UreF/UreH complex but interacts with the GST-UreF/UreH/urease complex.