Fig. 4.

Kinetics of AmyE and AmyL precursor processing, and BPN’ secretion in secDF, secG or dnaK mutant strains. Processing of AmyE or AmyL precursors (p) to the respective mature forms (m) was analyzed by pulse-chase labeling. Cells grown in MBU medium at 37 °C were labeled with [³⁵S]-methionine for 30 s prior to chase with excess non-radioactive methionine. Samples were withdrawn at the indicated time points after the chase and mixed with ice-cold TCA. Subsequently, (pre-)AmyE or (pre-)AmyL were immunoprecipitated with specific antibodies against AmyE or AmyL, separated by LDS-PAGE, and visualized by autoradiography. The secretion of BPN’ was also analyzed by pulse-chase labeling of cells grown in MBU at 37 °C with [³⁵S]-methionine for 30 s prior to chase with excess non-radioactive methionine. However, in this case, samples withdrawn at the indicated time points after the chase were chilled on ice and, subsequently, cells were separated from the growth medium by centrifugation. The appearance of BPN’ in the growth medium fractions was then analyzed by immunoprecipitation with antibodies against BPN’, LDS-PAGE and autoradiography. The position of mature BPN’ (m) is indicated