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Differential secretion of Dictyostelium discoideum proteinases
Abstract
Proteinases secreted by Dictyostelium discoideum myxamoebae during growth and starvation were analysed using three arginine-containing peptide nitroanilides as substrates. The enzymes were secreted differentially, both during growth and after resuspension of the myxamoebae in phosphate buffer. Secretion was stimulated by the addition of sucrose to starvation buffer, but this did not affect the type of proteinase released. Activity towards N-benzoyl-l-prolyl-l-phenylalanyl-l-arginine 4-nitroanilide (BzPFRase activity) was released in large quantities, and over 80% of the total activity in starved suspensions was extracellular within 6 h of transfer to buffer containing sucrose. A smaller fraction of the total activity towards N-carbobenzoxy-l-arginyl-l-arginine 4-nitroanilide (ZRRase activity) was secreted. Extracellular activity towards N-carbobenzoxy-l-tyrosyl-l-lysyl-l-arginine 4-nitroanilide (ZYKRase) was either too low to be detected or present in quantities which were small enough to be accounted for by cell lysis. BzPFRase and ZRRase activities were both inactivated by E-64 and two peptidyldiazomethanes, specific inhibitors of cysteine proteinases. ZYKRase activity was also inhibited by peptidyldiazomethanes but E-64 was effective only at high concentrations. When electrophoresis in polyacrylamide gels containing gelatin (gelatin-SDS-PAGE) was used to analyse the proteinases involved, one cysteine proteinase, ddCP42, correlated exactly with the secreted BzPFRase activity under all conditions, although contributions to this activity from other proteinases are also likely. Overall, the results demonstrate significant differences between individual proteinases with respect to secretion which suggests that their physiological roles may differ.
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© Society for General Microbiology, 1990