Purification and characterization of intracellular proteases of Clostridium perfringens type A.

Five intracellular proteases from sporulating cells of Clostridium perfringens type A were identified and three could be separated by DEAE-Sephacel. Two, I-A and I-B, had caseinolytic activity and one, I-C, was only active on N-benzoyl-DL-arginine-p-nitroanilide. I-A and I-B could each be further separated by Sephacryl S-300 into I-A-1 and I-A-2 and I-B-1 and I-B-2, respectively. I-A-1, a chymotrypsin-like enzyme, was the major intracellular protease, constituting 74% of the intracellular caseinolytic activity. In addition to cytoplasmic proteases both trypsin and chymotrypsin-like enzyme activity was associated with the membrane fraction. I-A-1 had a molecular weight of 330,000, with subunits of 120,000 and 138,000. I-A-1 cleaved a 1200 molecular weight peptide from C. perfringens enterotoxin. Early sporulating cell extracts of C. perfringens contained three presumptive enterotoxin precursors, which disappeared following treatment with I-A. Such cells also contained at least 10 spore coat related proteins, only one (51,500 molecular weight) of which was sensitive to I-A-1. The results indicate a possible role for the major intracellular protease in the processing of C. perfringens enterotoxin and a less important role, if any, in spore coat formation.