Schistosomes Enhance Plasminogen Activation: The Role of Tegumental Enolase (original) (raw)
Fig 5
Heterologous expression, purification and kinetics of r_Sm_Eno.
(A) Heterologous expression of r_Sm_Eno in E. coli BL21 Star (DE3). Coomassie-stained gel showing SDS-PAGE resolution of lysates of E. coli bacteria harboring pTrcHisB::_Sm_Eno before (Gel, left lane) or 4 hours after (Gel, right lane) protein expression induction. The arrow indicates r_Sm_Eno in the induced lane. In western blot analysis of these lysates probed with monoclonal anti-His tag antibody, a prominent ~50 kDa protein (_Sm_Eno) is detected (Western Blot, anti-His, arrow). Recombinant _Sm_Eno protein was purified from bacterial lysate by immobilized metal affinity chromatography. A single ~50 kDa pure protein (_Sm_Eno) is resolved following Coomassie brilliant blue staining of an SDS-PAGE gel and this protein binds anti-ENO antibody, as determined by western blot analysis (right lane, arrow). Positions of migration of molecular mass markers are indicated on the left (kDa). Michaelis-Menten kinetic curve generated using PGA as substrate (B, catalyzing the forward reaction) or using PEP as substrate (C, catalyzing the reverse reaction). The apparent Km and Vmax values shown represent the mean +/- SD of three independent experiments. (D) Recombinant _Sm_Eno activity in a buffer system covering the pH range 5.5–9.5. Enzymatic activity is maximal at pH 7.5. (E) Impact of divalent ion (Mg2+ or Ca2+, as indicated) concentration on mean r_Sm_Eno activity (± SD). The highest activity value (at 100 mM Mg2+) was set at 100% and relative activities were calculated and are presented. Significant differences relative to equivalent measurements containing Ca2+ are denoted by *** for p <0.001. EDTA is Ethylenediaminetetraacetic acid. (F) r_Sm_Eno activity in the presence of increasing concentrations of NaF (white bars) compared to its activity in the absence of inhibitors (set at 100%, black bar). Significant differences relative to the untreated control are denoted by *** for p <0.001. (G) Influence of increasing concentrations of mefloquine (MFQ) on r_Sm_Eno activity (left bars) or on schistosomula lysate enolase activity (right bars). Activity measured in the absence of MFQ was set at 100% and relative activities were calculated. Significant differences relative to the untreated control are denoted by * for p<0.05 and ** for p<0.01. In E-F, bars represent mean relative activity ± SD, n = 3.