Solubilization and initial biochemical characterization of an IgE-destroying enzyme on rat peritoneal mast cells.
Previous studies had demonstrated that incubation of IgE with purified rat mast cells can result in the time and cell concentration-dependent destruction of the ability of the IgE to be bound to specific receptors on rat basophil leukemia cells. The IgE-destroying activity, which has an extremely acid pH optimum, resisted attempts at solubilization using detergents. However, it was solubilized in good yield by use of chaotropic salts and especially KOCN. The soluble activity is stable to freezing and thawing, and to heating to 68 degrees C for 60 min. It is promptly destroyed upon boiling. IgE destruction was linear with time up to 20 min and a series of products, mol. wts 138,000, 92,500, 60,000 and 36,500, are formed during the reaction. No pH optimum for the reaction could be found because, as the pH was lowered below 4.0, the spontaneous destruction of IgE became too great. At pH 4.75 the apparent Km for the reaction was 0.55 microM and Vmax was 0.4 nmoles IgE/10(4) mast cell equivalents/min. IgE-destroying activity could be inhibited by heat-inactivated serum, and by relatively high concentrations of crude alpha 1-antitrypsin, aprotinin, lima bean and soybean trypsin inhibitors and by p-nitroguanidinobenzoate. A large number of other protease inhibitors were inactive.
D000818:Animals; D002478:Cells, Cultured; D006863:Hydrogen-Ion Concentration; D007073:Immunoglobulin E / Q000378:metabolism*; D007128:Immunoglobulin Fragments / Q000032:analysis; D008297:Male; D008407:Mast Cells / Q000201:enzymology*; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification / Q000378:metabolism*; D011480:Protease Inhibitors / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D007128:Immunoglobulin Fragments; D011480:Protease Inhibitors; D007073:Immunoglobulin E; C035774:IgE destroying enzyme; D010447:Peptide Hydrolases