TY - JOUR
T1 - Solubilization and initial biochemical characterization of an IgE-destroying enzyme on rat peritoneal mast cells
AU - Bach, Michael K.
AU - White, Gordon J.
AU - Johnson, Mark A.
AU - Ishizaka, Teruko
AU - Ishizaka, Kimishige
N1 - Funding Information:
*The work was supported in part by research grant AI-10060 from the U.S. Public Health Service and is publication No. 464 from the McNeil1 Laboratories of the Good Samaritan Hospital. t To whom correspondence snould he addressed at: Department of Hypersensitivity Diseases Research. Upjohn Co., Kalamazoo, MI 49001, U.S.A. jj Abbreviations: PCA, passive cutaneous anaphyiaxis; RBL-1. rat basophilic leukemia: BSA, bovine serum albumin; TPCK, I-tosylamide-2-phenylethyt chloromethylketone; TLCK, tosyl-L-lysine chloromethylketone: NPGB, p-nitroguanidinobenzoate; MES. 2-(N-morpholino)ethane sulfonic acid.
PY - 1982/8
Y1 - 1982/8
N2 - 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-dcslroying 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°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 μM and Vmaxwas 0.4nmoles IgE/104 mast cell equivalents/min. IgE-destroying activity could be inhibited by heat-inactivated serum, and by relatively high concentrations of crude α1-antitrypsin, aprotinin. limabean and soybean trypsin inhibitors and by p-nitroguanidinobenzoate. A large number of other protease inhibitors were inactive.
AB - 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-dcslroying 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°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 μM and Vmaxwas 0.4nmoles IgE/104 mast cell equivalents/min. IgE-destroying activity could be inhibited by heat-inactivated serum, and by relatively high concentrations of crude α1-antitrypsin, aprotinin. limabean and soybean trypsin inhibitors and by p-nitroguanidinobenzoate. A large number of other protease inhibitors were inactive.
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U2 - 10.1016/0161-5890(82)90307-8
DO - 10.1016/0161-5890(82)90307-8
M3 - Article
C2 - 6752697
AN - SCOPUS:0019970576
SN - 0161-5890
VL - 19
SP - 991
EP - 999
JO - Molecular Immunology
JF - Molecular Immunology
IS - 8
ER -