Amino acid sequence of rat mast cell protease I (chymase).
Amino acid sequence of rat mast cell protease I (chymase).
复制标题
大鼠肥大细胞蛋白酶 I（糜酶）的氨基酸序列。
DOI：
10.1021/bi00396a020
复制
发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Woodbury,RG
中科院分区：
文献类型：
--
作者：
LeTrong,H;Parmelee,DC;Walsh,KA;Neurath,H;Woodbury,RG
材料和方法大鼠肥大细胞蛋白酶I(RMCP I)的制备由Everett等人(1979)提出。JV-(/t-Tosyl)-L-苯丙氨酸-氯甲基酮-胰酶(TPCK-Trypsin)购自MilliPole。柠檬酸酐和BNPS-skatole购自皮尔斯化学公司。来自Achromobacter lyticus的赖氨酰内肽酶是日本茨城大学T.Masaki博士赠送的礼物。所有其他试剂和溶剂均以分析级或高效液相色谱级从商业上获得。还原和羧甲基化。在0.5M Tris-HCl、10 mM EDTA、45 mM二硫苏糖醇、0.5 M Tris-HCl和45 mM二硫苏糖醇的0.4 mL溶液中，室温下氮气孵育2 h，可还原RMCP I(120 Nmol)。然后加入新配制的100 mM碘乙酸溶液和100pl 1N NaOH溶液，在室温下黑暗中孵育30min。CMRMCP I采用反相高效液相色谱柱(Ul-tra孔RPSC-C3柱)纯化。RMCP的特异性切割是通过用TPCK-胰酶消化N-柠檬酰化的蛋白质来实现的。CM-RMCP I(20nmol)在0.5mL6-M盐酸胍中，加入16Pl柠檬酸酐，在室温下反应1h，用pH-盐保持pH为8.8，N-柠檬酰化(Atassi&Habeeb，1972)。然后用0.05M NH4HC03在pH 8.7的条件下对柠檬酰化的蛋白质进行广泛的透析。在4小时内分两份加入TPCK-胰酶
Materials and MethodsRat mast cell protease I (RMCP I) was prepared as de-scribed by Everett et al.(1979). jV-(/t-Tosyl)-L-phenylalanine chloromethyl ketone-trypsin (TPCK-trypsin) was purchased from Millipore. Citraconic anhydrideand BNPS-skatole were purchased from Pierce Chemical Co. Lysyl endopeptidase from Achromobacter lyticus was a gift from Dr. T. Masaki, Ibaraki University, Japan. All other reagents and solvents were commercially obtained as analytical or HPLC grades. Reduction and Carboxymethylation. RMCP I (120 nmol) was reduced by incubation under nitrogenfor 2 h at room temperature in 0.4 mL of 6 M guanidine hydrochloride 0.5 M Tris-HCl, 10 mM EDTA, and 45 mM dithiothreitol, pH 8.6. A freshly prepared solution of 100 mM iodoacetic acid in 100 pL of 1 N NaOH was then added, and the mixture was incubated in the dark at room temperature for 30 min. CMRMCP I was purified by reversed-phase HPLC on an Ul-trapore RPSC-C3 column (Altex). Specific Cleavage of RMCP I. Cleavage at arginyl residues was achieved by digesting N-citraconylated protein with TPCK-trypsin. CM-RMCP I (20 nmol) was N-citraconylated (Atassi & Habeeb, 1972) in 0.5 mL of 6 M guanidine hy-drochloride by addition of 16 pL of citraconic anhydride at room temperature over a period of 1 h while maintaining the pH at 8.8 with a pH-salt. The citraconylated protein was then extensively dialyzed against 0.05 M NH4HC03 at pH 8.7. TPCK-trypsin was added in two aliquots over a 4-h period at