After 30 min of labeling, cells were transferred to fresh phosphate-free MOPS medium containing 1 mM BSA and 150 μg/ml spectinomycin and allowed to incubate
further for 3 hr without CCCP. At 0 and 3 hr of chasing, equal volume of cell aliquot was withdrawn on ice, centrifuged and subjected to isolation of aggregated proteins. The isolated aggregates were immunoprecipitated with anti-AP antibody. The immunocomplex was run on 12% Gemcitabine concentration SDS-polyacrylamide gel, the gel was dried and subsequently set to autoradiography. The autoradiograph (Fig. 6B) of the electrophoresed immunoprecipitates indicated that the amount of AP-aggregate, after 3 hr of chasing (lane b), was about 66% INCB28060 in vivo less than its initial amount at 0 hr of chasing (lane a). This signified that the AP-aggregate had been degraded finally with time. It seemed that the degradation of AP-aggregate had been possibly caused by some induced heat-shock protease(s). When the degradation of the CCCP-mediated AP-aggregate
was checked, by the same ‘pulse-chase and immunoprecipitation’ experiment in two different E. coli mutants for the heat-shock proteases Lon (JT4000) and ClpP (SG22159), it was observed that in the clpP mutant, no degradation of the AP-aggregate took place (lanes c and d, Fig. 6B); whereas in the lon mutant, degradation occurred (lanes e and f, Fig. 6B). This result clearly implied that not the major heat-shock www.selleck.co.jp/products/Gemcitabine(Gemzar).html protease Lon, rather a minor protease ClpP was responsible for the degradation P505-15 nmr phenomenon. Such degradation removed the translocation-incompetent,
non-functional AP and thus was essential for cell survival; this was supplemented from the fact that the clpP mutant (SG22159) was more sensitive to CCCP than wild type strain SG20250. In the presence of 25 μM CCCP, where the wild type cells had some growth, the mutant cells became bacteriostatic, and by the treatment of 50 μM CCCP for 90 min, where there was no killing of E. coli SG20250 cells, about 90% cell-killing occurred in case of E. coli SG22159 strain (data not shown). When the cell extract of AP-induced culture was subjected to two-step immunoprecipitation study using anti-DnaK and anti-AP antibodies serially, the final immunoprecipitate of the CCCP-treated cells, in contrast to that of the control cells, had contained AP in addition to the DnaK protein (fig. 7A). This clearly signified that the first immunoprecipitate with anti-DnaK antibody had certainly contained AP i.e., the non-translocated AP in the CCCP-treated cells was present in cell cytosol as a binary complex form with DnaK. This result justified the fact of sigma-32 stabilization in the protonophores-treated cells as – the non-translocated proteins had signaled DnaK/J to bind with them, finally freeing and so stabilizing sigma-32.