Rat Skin Main Neutral Protease: Purification and Properties

Abstract

The main neutral protease of rat skin has been purified by extracting the skin first with a dilute buffer, solubilizing the enzyme with 2M KCl, precipitating it by dialysis in dilute buffer, resolubilizing the neutral protease with 2M KCl, chromatographying it on Sephadex G-200 and hydroxyapatite. The purified enzyme is homogenous in sodium dodecylsulfate-gel electrophoresis. It is cationic and, at low ionic strength, forms complexes with heparin, is adsorbed by dextran and polyacrylamide gels, and glass. A molecular weight of 27,500 was obtained with sodium dodecylsulfate-gel electrophoresis and gel chromatography. The specificity of the enzyme resembles that of bovine chymotrypsin; it hydrolyzes proteins and synthetic substrates of chymotrypsin, optimally at pH 8–9. The enzyme is activated by KCl and inhibited by diisopropyl fluorophosphate, phenylmethyl sulfonyifluoride, tosylphenylalanine chloromethylketone, trypsin inhibitors from lima bean and soybean, and by rat serum. The trypsin inhibitors from ovomucoid or bovine lung (Trasylol) did not inhibit the enzyme. Treatment of rats with compound 48/80, a mast cell degranulator, decreases the amount of the neutral protease in skin to 1/50 of the control. This, and the properties of the enzyme, suggest that the main neutral protease of rat skin is identical with the rat mast cell chymase.