Shivaraj B, Pattabiraman T N
Biochem J. 1981 Jan 1;193(1):29-36. doi: 10.1042/bj1930029.
An inhibitor I-1, capable of acting on both alpha-amylase and trypsin, was purified to homogeneity from ragi (finger-millet) grains. The factor was found to be stable to heat treatment at 100 degrees C for 1 h in the presence of NaCl and also was stable over the wide pH range 1-10. Pepsin and Pronase treatment of inhibitor I-1 resulted in gradual loss of both the inhibitory activities. Formation of trypsin-inhibitor I-1 complex, amylase-inhibitor I-1 complex and trypsin-inhibitor I-1-amylase trimer complex was demonstrated by chromatography on a Bio-Gel P-200 column. This indicated that the inhibitor is 'double-headed' in nature. The inhibitor was retained on a trypsin-Sepharose 4B column at pH 7.0. Elution at acidic pH resulted in almost complete recovery of amylase-inhibitory and trypsin-inhibitory activities. alpha-Amylase was retained on a trypsin-Sepharose column to which inhibitor I-1 was bound, but not on trypsin-Sepharose alone. Modification of amino groups of the inhibitor with 2,4,6-trinitrobenzenesulphonic acid resulted in complete loss of amylase-inhibitory activity but only 40% loss in antitryptic activity. Modification of arginine residues by cyclohexane-1,2-dione led to 85% loss of antitryptic activity after 5 h, but no effect on amylase-inhibitory activity. The results show that a single bifunctional protein factor is responsible for both amylase-inhibitory and trypsin-inhibitory activities with two different reactive sites.
一种能够作用于α-淀粉酶和胰蛋白酶的抑制剂I-1，从龙爪稷（黍）种子中纯化至同质。该因子在NaCl存在下于100℃热处理1小时后仍保持稳定，并且在1-10的宽pH范围内也很稳定。用胃蛋白酶和链霉蛋白酶处理抑制剂I-1会导致两种抑制活性逐渐丧失。通过在Bio-Gel P-200柱上进行色谱分析，证明了胰蛋白酶-抑制剂I-1复合物、淀粉酶-抑制剂I-1复合物和胰蛋白酶-抑制剂I-1-淀粉酶三聚体复合物的形成。这表明该抑制剂本质上是“双头”的。该抑制剂在pH 7.0时保留在胰蛋白酶-琼脂糖4B柱上。在酸性pH下洗脱几乎完全恢复了淀粉酶抑制活性和胰蛋白酶抑制活性。α-淀粉酶保留在结合了抑制剂I-1的胰蛋白酶-琼脂糖柱上，但单独的胰蛋白酶-琼脂糖柱上则没有。用2,4,6-三硝基苯磺酸修饰抑制剂的氨基会导致淀粉酶抑制活性完全丧失，但抗胰蛋白酶活性仅丧失40%。用环己烷-1,2-二酮修饰精氨酸残基5小时后导致抗胰蛋白酶活性丧失85%，但对淀粉酶抑制活性没有影响。结果表明，一个单一的双功能蛋白质因子负责具有两个不同反应位点的淀粉酶抑制活性和胰蛋白酶抑制活性。
