Sakal E, Applebaum S W, Birk Y
Department of Biochemistry, Faculty of Agriculture, Hebrew University of Jerusalem, Rehovot, Isreal.
Int J Pept Protein Res. 1988 Dec;32(6):590-8. doi: 10.1111/j.1399-3011.1988.tb01391.x.
A chymotrypsin-like enzyme (CTLE) was isolated from the digestive tract of the African migratory locust Locusta migratoria migratorioides by ion-exchange chromatography on diethylaminoethyl (DEAE) cellulose followed by affinity chromatography on phenylbutylamine (PBA) Sepharose. The purity and homogeneity of CTLE have been shown by SDS-PAGE and on cellulose acetate strips. The enzyme has a molecular weight of 24,000, determined by SDS-PAGE and on a Sephadex G-75 calibrated column. It has an isoelectric point of 10.1 and contains 0-1 half cystine residues. Sequence analysis of the first 20 N-terminal amino acids has shown 25% homology with bovine chymotrypsin and 40% homology with Vespa crabo and Vespa orientalis chymotrypsins and with Hypoderma lineatum trypsin. The optimal pH for enzyme activity and stability was in the range of 8.5-9.0. The Km and kcat values, determined on substrates for proteolytic, esterolytic and amidolytic activity, similar to those for bovine chymotrypsin. CTLE was inactivated by PMSF and TPCK indicating the involvement of serine and histidine in its active site. The enzyme was fully inhibited by the proteinaceous, double-headed, chymotrypsin-trypsin inhibitors BBI from soybeans and CI from chickpeas, by chicken ovomucoid (COM) and turkey ovomucoid (TOM), as well as by the Kunitz soybean trypsin inhibitor (STI) which hardly inhibits bovine chymotrypsin. Inhibition studies of CTLE with amino acid and peptide-chloromethylketones point towards the existence of an extended binding site.
通过在二乙氨基乙基（DEAE）纤维素上进行离子交换色谱，随后在苯基丁胺（PBA）琼脂糖上进行亲和色谱，从非洲迁徙蝗虫（Locusta migratoria migratorioides）的消化道中分离出一种类胰凝乳蛋白酶（CTLE）。CTLE的纯度和均一性已通过SDS-PAGE和醋酸纤维素条带得以证明。通过SDS-PAGE和在经校准的葡聚糖G-75柱上测定，该酶的分子量为24,000。其等电点为10.1，含有0至1个半胱氨酸残基。对前20个N端氨基酸的序列分析表明，它与牛胰凝乳蛋白酶有25%的同源性，与黄蜂（Vespa crabo）和东方黄蜂（Vespa orientalis）的胰凝乳蛋白酶以及纹皮蝇（Hypoderma lineatum）胰蛋白酶有40%的同源性。该酶活性和稳定性的最佳pH范围为8.5至9.0。在蛋白水解、酯水解和酰胺水解活性底物上测定的Km和kcat值与牛胰凝乳蛋白酶的相似。CTLE被PMSF和TPCK灭活，表明丝氨酸和组氨酸参与其活性位点。该酶被来自大豆的蛋白质类双头胰凝乳蛋白酶-胰蛋白酶抑制剂BBI、来自鹰嘴豆的CI、鸡卵类粘蛋白（COM）和火鸡卵类粘蛋白（TOM）以及几乎不抑制牛胰凝乳蛋白酶的Kunitz大豆胰蛋白酶抑制剂（STI）完全抑制。用氨基酸和肽氯甲基酮对CTLE进行的抑制研究表明存在一个扩展的结合位点。
