Intracellular serine protease was isolated from stationary-grown Bacillus subtilis A-50 cells and purified to homogeneity. The molecular weight of the enzyme is 31,000 ± 1,000, with an isoelectric point of 4.3. Its amino acid composition is characteristically enriched in glutamic acid content, differing from that of extracellular subtilisins. The enzyme is completely inhibited with phenylmethylsulfonyl fluoride and ethylenediaminetetraacetic acid. Intracellular protease possesses negligible activity towards bovine serum albumin and hemoglobin, but has 5to 20-fold higher specific activity against p-nitroanilides of benzyloxycarbonyl tripeptides than subtilisin BPN'. Esterolytic activity of the enzyme is also higher than that of subtilisin BPN'. The enzyme is sequence homologous with secretory subtilisins throughout 50 determined NH2-terminal residues, indicating the presence of duplicated structural genes for serine proteases in the B. subtilis genome. The occurrence of two homologous genes in the cell might accelerate the evolution of serine protease not only by the loosening of selective constraints, but also by creation of sequence variants by means of intragenic recombination. Three molecular forms of intracellular protease were found, two of them with NH2terminal glutamic acid and one minor form, three residues longer, with asparagine as NH2 terminus. These data indicate the possible presence of an enzyme precursor proteolytically modified during cell growth.
Intracellular serine proteinase was isolated from sporulating cells of Bacillus subtilis Marburg 168 by gramicidin S-Sepharose 4B affinity chromatography. The enzymological characteristics, the amino acid composition and the 19 residues of the N-terminal sequence of the enzyme are reported. The isolated proteinase was closely related to, but not completely identical with, the intracellular serine proteinase of B. subtilis A-50. The divergence between these two intracellular enzymes was less than that between the corresponding extracellular serine proteinases (subtilisins) of types Carlsberg and BPN', produced by these bacterial strains. This may be connected with the more strict selection constraints imposed in intracellular enzymes during evolution.
While about 80% of the cell-bound intracellular serine protease of Bacillus subtilis A-50 have been recovered in the soluble fraction upon disruption of cells, the rest of the enzyme was found to be associated with the membrane fraction. Soluble cytoplasmic intracellular serine protease, as well as membrane-bound serine protease liberated by non-ionic detergent treatment, have been isolated in a pure state and shown to be identical. The same protease might also be found extracellularly, due presumably to cell lysis or altered membrane permeability. Intracellular serine protease of Bacillus subtilis A-50 was clearly related to Bacillus subtilis serine proteases W1 and bacillopeptidase F described as extracellular enzymes.
Two serine proteases in extracts of Escherichia coli grown to stationary phase were purified to homogeneity using affinity chromatography on gramicidin S-Sepharose 4B. One enzyme was closely related to, if not identical with, the ' trypsin-like' protease I1 of E. coli. The other was capable of cleaving the subtilisin chromogenic substrate N-carbobenzoxy-L-alanyl-Lalanyl-L-leucine-p-nitroanilide and resembled the intracellular serine proteases of Bacillus spp. The amino acid composition of this E. coli protease was similar to that of the Bacillus lichenformis enzyme. These data indicate a relationship between proteolytic enzymes of evolutionary distant Gram-negative Enterobacteriaceae and Gram-positive spore-forming Bacillus.
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