Detection of the Endosomal Sorting Complex Required for Transport in Entamoeba histolytica and Characterization of the EhVps4 Protein
Figure 4
Measurement of ATPase activity of recombinant EhVps4. (a) Multiple sequence alignment of Vps4 AAA domain from Homo sapiens (HsVps4A and HsVps4B), S. cerevisiae (ScVps4p) and E. histolytica (EhVps4). Black boxes, identical amino acid (aa); grey boxes, conserved substitutions; open boxes, different aa. Numbers at the left are relative to the position of the initial methionine in each protein. Arrowhead indicates the position of the conserved E residue. (b) Purification of recombinant proteins. Proteins were expressed in E. coli, purified through affinity chromatography, separated through 10% SDS-PAGE and stained with Coomassie blue. Lane 1, rEhVps4-GST; lane 2, rEhVps4-E211Q-GST; lane 3, rGST; lane 4, purified fraction from noninduced bacteria extract. (c) ATPase assay. ATPase activity of rEhVps4-GST and rEhVps4-E211Q-GST was monitored as described in Section 2.7. rGST was included as control. Data are the mean of three independent assays.
