Characterization of a neutral protease from lysosomes of rabbit polymorphonuclear leucocytes.
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.
D000614:Aminocaproates / Q000494:pharmacology; D000818:Animals; D001202:Ascitic Fluid / Q000166:cytology; D002501:Centrifugation, Zonal; D002850:Chromatography, Gel; D003593:Cytoplasm; D003911:Dextrans / Q000494:pharmacology; D006454:Hemoglobins / Q000378:metabolism; D006493:Heparin / Q000494:pharmacology; D006657:Histones / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007962:Leukocytes / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D010447:Peptide Hydrolases / Q000032:analysis* / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011480:Protease Inhibitors; D011817:Rabbits; D013347:Subcellular Fractions; D013431:Sulfates / Q000494:pharmacology; D013449:Sulfonamides / Q000494:pharmacology; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000614:Aminocaproates; D003911:Dextrans; D006454:Hemoglobins; D006657:Histones; D011480:Protease Inhibitors; D013431:Sulfates; D013449:Sulfonamides; D014361:Trypsin Inhibitors; D011189:Potassium Chloride; D006493:Heparin; D010447:Peptide Hydrolases