Saito N
J Biol Chem. 1982 Mar 25;257(6):3120-5.
A strain of fungus Cladosporium herbarum extracellularly produced an inhibitor specific for mammalian alpha-amylase. The inhibitor was purified 81-fold by freeze-thawing, heat treatment, and column chromatography on DEAE-cellulose, Sephadex G-75, DEAE-Sephacel, and Bio-Gel P-100. An apparent molecular weight of approximately 18,000 was estimated for the inhibitor using Bio-Gel P-100 filtration. The purified inhibitor preparation was a glycoprotein containing about 10% carbohydrate. The amino acid analysis of the inhibitor showed abundances of Gly, Asp, Glu, Ser, Ala, and Thr residues. The inhibitor was stable between pH 5 and 12 at 4 degrees C, and below 80 degrees C at pH 7.0. A binary complex formation out of equimolar amounts of the inhibitor and alpha-amylase, was demonstrated by polyacrylamide gel electrophoresis, and Bio-Gel P-100 chromatography. Kinetic studies exhibited that the inhibitor noncompetitively inhibited the enzyme reaction with a Ki value of 2.3 approximately 4.8 x 10(-10) M, by combining with the enzyme molecule at a different site from the substrate binding site.
一株草本枝孢菌能在细胞外产生一种对哺乳动物α-淀粉酶具有特异性的抑制剂。通过冻融、热处理以及在DEAE-纤维素、葡聚糖G-75、DEAE-琼脂糖凝胶和生物凝胶P-100上进行柱色谱，该抑制剂被纯化了81倍。使用生物凝胶P-100过滤法估计该抑制剂的表观分子量约为18,000。纯化后的抑制剂制剂是一种糖蛋白，含有约10%的碳水化合物。对该抑制剂的氨基酸分析表明，甘氨酸、天冬氨酸、谷氨酸、丝氨酸、丙氨酸和苏氨酸残基含量丰富。该抑制剂在4℃下pH值为5至12之间稳定，在pH 7.0时80℃以下稳定。通过聚丙烯酰胺凝胶电泳和生物凝胶P-100色谱证明，等摩尔量的抑制剂和α-淀粉酶形成了二元复合物。动力学研究表明，该抑制剂通过与酶分子上不同于底物结合位点的位点结合，以非竞争性方式抑制酶反应，Ki值约为2.3至4.8×10⁻¹⁰ M。
