AP was known to be synthesized initially in the cytoplasm and then translocated out through the inner membrane to be finally localized as dimeric, active form at the periplasm [32, 33]. As the dimerization of AP, through the disulfide bond, could not take

GSK690693 nmr place in the reducing milieu of the cytoplasmic environment, the cytosolic pool of the nontranslocated AP in the CCCP-treated cells had shown no activity [34, 35]. Figure 4 A. L evel of active AP in E. coli MPh42 cells grown in the presence of different concentrations of CCCP. Cells were initially grown to log phase (~1.5 × 108 cells/ml) at 30°C in complete MOPS medium and were then transferred to phosphate-less MOPS medium. The re-suspended cells were divided in different parts to treat with the different concentrations of CCCP (0, 10, 30 and 50 μM). The divided

cell cultures were then allowed to grow further at 30°C for induction of AP. At different intervals of time, a 1.0 ml cell aliquot was withdrawn from each culture to assay the active AP level. B. Western blot of the different fractions (periplasmic, cytoplasmic and membrane) PF-6463922 datasheet of E. coli MPh42 cells grown in the presence of CCCP (50 μM). After allowing induction of AP for 30 min, the periplasmic, cytoplasmic and membrane fractions were isolated from equal number of each of the CCCP-treated and the control cells and the western blotting experiment was subsequently performed using anti-AP antibody. Lanes (a, b, c) and (e, f, g) represent the membrane, periplasmic and cytoplasmic fractions of control and CCCP-treated cells respectively; lane d represents purified AP. To investigate whether the non-translocated AP in cell cytosol could have been transported out to the periplasm on withdrawal of CCCP from the growth medium, pulse-chase and immunoprecipitation experiment was performed. Cells, grown in phosphate-free (required for the induction of AP) and

methionine-free MOPS medium in the presence of 50 μM CCCP, were radio-labeled with 35S-methionine for 30 min; the CCCP was then removed IMP dehydrogenase by centrifugation and the cells were resuspended in the phosphate-less MOPS medium. Finally the chasing with cold methionine was allowed for 1 hr. The periplasmic fractions of the chased cells were isolated, immunoprecipitated with anti-AP antibody, the immunoprecipitates were run in 12% SDS-polyacrylamide gel, western blotting with anti-AP antibody was done and the blotted membrane was finally autoradiographed [36]. The autoradiograph (Fig. 5A) showed that the periplasmic fraction of the CCCP-treated cells had contained no trace of AP (lane b), whereas that of the control cells contained it (lane a). This signified that the AP, synthesized during the presence of CCCP (i.e., for the labeling period of 30 min), could not be translocated out to the periplasm, even after 60 min of chasing in the absence of CCCP. The western blot result (Fig.