Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes

Abstract

1. The subcellular distribution has been investigated of a protease from rabbit polymorphonuclear leucocytes, obtained from peritoneal exudates. The enzyme, optimally active between pH7.0 and 7.5, hydrolyses histone but not haemoglobin, sediments almost exclusively with a granule fraction rich in other lysosomal enzymes, and is latent until the granules are disrupted by various means. 2. Enzymic analysis of specific and azurophilic granules separated by zonal centrifugation showed that neutral protease activity was confined to fractions rich in enzymes characteristic of azurophile granules. 3. Recovery of neutral protease activity from subcellular fractions was several times greater than that found in whole cells. This finding was explained by the presence of a potent inhibitor of the enzyme activity in the cytoplasm. 4. The effect of the inhibitor was reversed by increasing ionic strength (up to 2.5m-potassium chloride) and by polyanions such as heparin and dextran sulphate, but not by an uncharged polymer, dextran. 5. The enzyme was also inhibited, to a lesser extent, by 1-chloro-4-phenyl-3-l-toluene-p-sulphonamidobutan-2-one, soya-bean trypsin inhibitor and in-aminohexanoate (in-aminocaproate). 6. The granule fractions failed to hydrolyse artificial substrates for trypsin and chymotrypsin. 7. Partial separation of the enzyme was achieved by Sephadex gel filtration at high ionic strength and by isoelectric focusing. The partially separated, activated enzyme showed an approximately 300-fold increase in specific activity over that in whole cells.

Proteoglycan-degrading enzymes of rabbit fibroblasts and granulocytes

“…By contrast, the proteolytic activity of rabbit polymorphonuclear leucocytes is mainly at acid pH values (Davies et al, 1971;Ignarro et al, 1973)andtheproteoglycan-degradingactivityhasbeen found to show a similar characteristic (Weissmann & Spilberg, 1968;Oronsky et al, 1973). It was therefore surprising that we found greater activity at pH7.6 than at pH4.5 in the bead assay for proteoglycan-degrading activity.…”

Intracellular Distribution of Neutral Proteinases and Inhibitors in Pig Leucocytes. Isolation of Two Inhibitors of Neutral Proteinases

“…In Davies' [7] opinion, who obtained leucocytes by glycogen induction, the inhibitor uptake from plasma is possible.…”

“…Davies and coworkers [7] reported in their study of neutral proteinases of polymorphonuclear cells that cytoplasmic -postgranular supernatant fraction caused the strongest inhibition of neutral proteinases. Because the polymorphonuclear cells were obtained from inflammatory exudates, they proposed a possibility of inhibitor uptake from plasma.…”

Substrate specificity and modifications of the active centre of elastase-like neutral proteinases from horse blood leucocytes

“…elastase-like enzymes, in distinction to human leucocytes (Ohlsson & Olsson, 1973Schmidt & Havemann, 1974), Hence postulated species-dependent differences in the enzymic pattern of leucocyte neutral proteinases (Davies et al, 1971;Dewald et al, 1975) seem to be confirmed.…”