PURIFICATION AND CHARACTERIZATION OF SERINE PROTEINASE-INHIBITORS FROM GOURD (LAGENARIA-LEUCANTHA RUSBY VAR GOURDA MAKINO) SEEDS
PURIFICATION AND CHARACTERIZATION OF SERINE PROTEINASE-INHIBITORS FROM GOURD (LAGENARIA-LEUCANTHA RUSBY VAR GOURDA MAKINO) SEEDS
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DOI：
10.1271/bbb.56.275
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发表时间：
1992-02-01
影响因子：
1.6
通讯作者：
HARA, S
中科院分区：
文献类型：
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作者：
HAMATO, N;TAKANO, R;HARA, S
葫芦种子抑制剂以下列方式纯化：将葫芦种子研磨并用10 mM碳酸铵（pH 7.8）提取。用65-90%丙酮沉淀提取物，并将丙酮沉淀物在Cellulofine GCL-90-m柱中凝胶过滤。将显示胰蛋白酶抑制活性的3000 Da级分合并，并通过离子交换和反相色谱进一步纯化，从而将三种抑制剂LLTI-I、II和III纯化至均一，这些抑制剂的氨基酸序列为：[图表]确切序列是独特的，但与属于南瓜家族的蛋白酶抑制剂非常相似。基于序列，假设在三种抑制剂中发现的肽键（Arg-Ile）是胰蛋白酶的反应位点。LLTI-I、II和III与牛胰蛋白酶复合物的Ki值估计值分别为3.6 x 10（-10）M、6.5 x 10（-11）M和3.0 x 10（-11）M。
Gourd seed inhibitors were purified in the following manner: gourd seeds were ground and extracted with 10 mM ammonium carbonate, pH 7.8. The extract was precipitated with 65-90% acetone and the acetone precipitates were gel filtered in a Cellulofine GCL-90-m column. Fractions of 3000 Da showing trypsin inhibitory activity were combined and purified further by ion exchange and reversed phase chromatographies.Three inhibitors, LLTI-I, II, and III were thus purified to homogeneity and the amino acid sequences of these inhibitors were:[GRAPHICS]The exact sequences are unique but very similar to proteinase inhibitors belonging to the squash family. Based on the sequence, it is assumed that the peptide bond (Arg-Ile) found in the three inhibitors is the reactive site for trypsin. The Ki values estimated for complexes of LLTI-I, II, and III with bovine trypsin were 3.6 x 10(-10) M, 6.5 x 10(-11) M, and 3.0 x 10(-11) M, respectively.