Some properties of neutral proteinases from lysosomes of rabbit polymorphonuclear leukocytes

Neutral proteinases capable of degrading proteoglycan were found in lysosomes of rabbit polymorphonuclear leucocytes extracted with 0.01 M citric acid. Esterase activity against an elastase substrate was also present but chymotrypsin- and trypsin-like activities were not detected; azocasein-degrading activity was poor. Proteoglycanase activity was stimulated by high concentrations of salts (0.2 M KCl) and divalent cations (Ca, Mg, Mn, Zn) but was inhibited by Cu ++ . Elastase activity was also stimulated by high ionic strength buffers and KCl, but not as much by divalent cations, and was inhibited by Cu ++ . Proteoglycanase in crude extracts was inhibited by EDTA, phenylmethanesulphonylfluoride (Pms-F), cell cytosol, α 1 -antitrypsin, gold thiomalate and N-acetyl-di-L-alanyl-L-prolyl-L-valine chloromethyl ketone (AAAPVCK). Partial inhibition by N-α-p-tosyl-L-lysine chloromethyl ketone (TLCK) and L-1-tosylamide-2-phenylethyl chloromethyl ketone (TPCK) occurred. Elastase adsorbed to CM-cellulose and was eluted by 0.6-0.7 M NaCl; a metallo-proteinase failed to adsorb completely but was retarded by the CM-cellulose. Isoelectric focusing showed that the major proteinases had pI's of 5.5, 8.5 and 9.1; the activity with pI 8.5 was a metalloproteinase, and the pI 9.1 activity was an elastase. The apparent molecular weight of the elastase, determined on Sephadex G-100, was 8,000 to 11,000 daltons.