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SOME PROPERTIES OF NEUTRAL PROTEINASES FROM LYSOSOMES OF RABBIT POLYMORPHONUCLEAR LEUCOCYTES
Author(s) -
Britz ML,
Lowther DA
Publication year - 1981
Publication title -
australian journal of experimental biology and medical science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.999
H-Index - 104
eISSN - 1440-1711
pISSN - 0004-945X
DOI - 10.1038/icb.1981.3
Subject(s) - chemistry , elastase , divalent , chymotrypsin , isoelectric point , trypsin , biochemistry , ionic strength , enzyme , chromatography , organic chemistry , aqueous solution
Summary 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 metallo‐proteinase, 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.