Isolation And Some Properties Of A Proteolytic Enzyme From Escherichia-Coli (Protease I)

By the use of electrophoretic and spectrophotometric methods two types of endopeptidases were demonstrated in crude extracts of Escherichia coli: one was active on N-acetyl-dl-phenyl-alanine-2-naphtyl ester and the other on N-benzoyl-l-arginine-p-nitroanilide. The two activities were separated by gel filtration on Sephadex G-100. After gel electrophoresis of crude extracts, three bands of activity toward acetyl-phenylalanine-naphthyl ester were visualized. The enzyme corresponding to the band with the strongest esterolytic activity was also responsible for casein-hydrolytic activity in gel between pH 6 and 8; it was isolated and characterized. Starting with 450 g of wet cells, about 3 mg of a purified enzyme were obtained. This represented a recovery of 16% and almost 1000-fold purification. The isolated enzyme, designated as protease I, was homogeneous by electrophoresis and sucrose-gradient centrifugation. Its molecular weight was estimated by two different methods to be about 43000. The enzyme hydrolysed N-acetyl-dl-phenylalanine-2-naphthyl ester, a chymotrypsin substrate, but was inactive upon several synthetic substrates for enzymes with carboxypeptidase-A and trypsin-like specificity. The esterolytic activity was inhibited by DFP, whereas it was resistant to phenyl methyl-sulfonylfluoride even after prolonged treatment. Metal chelating agents, sulfhydryl reagents and several metal ions were without effect. The proterolytic activity of the purified enzyme was confirmed by its ability to breakdown native E. coli polynucleotide phosphorylase.