BacS and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as
proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R.,
Horstmann, H. and Romeo, D. (1990) J. Cell Bid. I l l , 1363-13711. These proforms have been
purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography.
While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella
typhimurium with minimal inhibitory concentrations of 6- 12 pg/ml, proBac5 and proBac7 do not
affect the growth of the same microorganisms, even at 500 pg/ml. Previous investigations have
suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a
neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus
treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine
proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass
spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to
catalyze the stepwise cleavage of probactenecins into the corresponding mature fieptides, which have
the same mass, N-terminal sequence and antibiotic activity of authentic BacS and Bac7. These results
point to the importance of cooperation between azurophils and large granules in mounting a defense
reaction.
