Differential in vitro activation and deactivation of cysteine proteinases isolated during spore germination and vegetative growth of Dictyostelium discoideum

Acid-activatable cysteine proteinases of Dictyostelium discoideum were firs t identified in spore extracts of strain SG1 using gelatin/SDS/PAGE, follow ed by acid treatments. Here we utilized the technique of acid activation to identify cryptic cysteine proteinases throughout auto-induced and heat-ind uced spore germination of D. discoideum strain SG2 and SG1. The major acid- activatable cysteine proteinase identified in SG2 and SG1 spore extracts wa s ddCP38 (D. discoideum cysteine proteinase with a molecular mass of 38 kDa ) and ddCP48, respectively. Further investigation of these enzymes revealed that they were also base deactivatable with a treatment of ammonium chlori de directly following acid activation. However, the most intriguing observa tion was the reversibility of the effects of base deactivation on the enzym es following a second treatment with acetic acid. Thus, we hypothesize that , unlike most mammalian cysteine proteinases which generally require the cl eavage of a pro-peptide region for activation, these cysteine proteinases o f D. discoideum likely undergo reversible conformational changes between la tent and active forms. Moreover, we were able to detect these cryptic cyste ine proteinases in the vegetative cells and early aggregates of both strain s SG1 and SG2. Studies using 4-[(2S,3S)-3-carboxyoxiran-7-ylcarbonyl-L-leuc ylamido]butylguanidine, a cysteine proteinase inhibitor, revealed that acid activation of a portion of these proteinases was still achievable even aft er incubation with the inhibitor further supporting the concept of two stab le and reversible conformational arrangements of the enzymes. Thus, we spec ulate that the pH shuffles that modulate proteinase conformation and activi ty in vitro may be a reflection of the in vivo regulation of these enzymes via H+-ATPases and ammonia.