SOME PROPERTIES OF NEUTRAL PROTEINASES FROM LYSOSOMES OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES

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Abstract
Neutral proteinases capable of degrading proteoglycan were found in lysosomes of rabbit polymorphonuclear leucocytes extracted with 001 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 (02 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, 1-antitrypsin, gold thiomalate and N-acetyl-di-L-alanyl-L-prolyl-L-valine chloromethyl ketone (AAAPVCK). Partial inhibition by N--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 06-07 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 55, 85 and 91; the activity with pI 85 was a metallo-proteinase, and the pI 91 activity was an elastase. The apparent molecular weight of the elastase, determined on Sephadex G-100, was 8,000 to 11,000 daltons.