Possible role of calcium in the formation of active extracellular proteinase by Pseudomonas fluorescens

The requirement for calcium during synthesis of extracellular proteinase by Pseudomonas fluorescens B52 was examined. Synthesis was monitored using cells resuspended at high density in fresh growth medium. Optimum enzyme production was found with cells grown to mid-logarithmic phase in mineral salts medium containing calcium chloride (1.0 mmol/l). Inhibition of synthesis by EDTA addition was rapid, similar to the effect produced by chloramphenicol, an inhibitor of translation. Appearance of enzyme initiated by calcium addition to depleted cells was rapid and was dependent on de novo protein synthesis. Sephadex G-100 chromatography of L-[4,5-3H]leucine-labelled cell-free supernatant liquids revealed that, in the absence of calcium, a low molecular weight (12000-14000 daltons) irreversibly inactive 'precursor' of the proteinase was formed. The results are consistent with the hypothesis that calcium is required for structural integrity of the proteinase as well as for activity.