Ishikawa C, Watanabe K, Sakata N, Nakagaki C, Nakamura S, Takahashi K
J Biochem. 1985 Jan;97(1):55-70. doi: 10.1093/oxfordjournals.jbchem.a135068.
Double-headed protease inhibitors I, IIa, and IIc (AB I, AB IIa, and AB IIc) have been purified from azuki beans "Takara" (Vigna angularis) by conventional chromatographic methods and their amino acid sequences have been determined. AB I, AB IIa, and AB IIc had molecular weights of 9,166, 8,661, and 8,756 daltons, consisting of 82, 78, 79 amino acid residues, respectively. The molecular weights of these inhibitors, determined by gel filtration at pH 8.0, were 18,000 for AB I and 17,000 for both AB IIa and AB IIc, indicating that the inhibitors are dimers. The inhibitors had isoelectric points of 4.7 (AB I), 6.8 (AB IIa), and 6.2 (AB IIc). AB I stoichiometrically inhibited both trypsin and chymotrypsin at a molar ratio of 1 : 1. On the other hand, AB IIa and AB IIc both inhibited trypsin at a molar ratio of about 1 : 2 and also inhibited chymotrypsin, though only weakly. Sequence comparison with other double-headed inhibitors indicated the reactive sites of AB IIa and AB IIc for trypsin to be Lys26-Ser27 and Arg53-Ser54, and those of AB I for trypsin and chymotrypsin to be Lys26-Ser27 and Tyr53-Ser54, respectively. The differences between AB IIa and AB IIc were that AB IIa lacked the C-terminal aspartic acid residue, and that Glu10 and Arg60 in AB IIa were replaced by Gln10 and His60 in AB IIc. A comparison between AB IIa and AB I revealed 25 variant amino acids among the 78 residues of AB IIa; further, Ab IIa lacked 4 amino acid residues in the C-terminal region of AB I.
通过传统色谱方法从“Takara”小豆（Vigna angularis）中纯化出双头蛋白酶抑制剂I、IIa和IIc（AB I、AB IIa和AB IIc），并测定了它们的氨基酸序列。AB I、AB IIa和AB IIc的分子量分别为9166、8661和8756道尔顿，分别由82、78、79个氨基酸残基组成。在pH 8.0条件下通过凝胶过滤测定，这些抑制剂的分子量AB I为18000，AB IIa和AB IIc均为17000，表明这些抑制剂是二聚体。这些抑制剂的等电点分别为4.7（AB I）、6.8（AB IIa）和6.2（AB IIc）。AB I以1:1的摩尔比化学计量地抑制胰蛋白酶和胰凝乳蛋白酶。另一方面，AB IIa和AB IIc均以约1:2的摩尔比抑制胰蛋白酶，并且也抑制胰凝乳蛋白酶，尽管抑制作用较弱。与其他双头抑制剂的序列比较表明，AB IIa和AB IIc对胰蛋白酶的反应位点分别为Lys26 - Ser27和Arg53 - Ser54，AB I对胰蛋白酶和胰凝乳蛋白酶的反应位点分别为Lys26 - Ser27和Tyr53 - Ser54。AB IIa和AB IIc之间的差异在于AB IIa缺少C末端天冬氨酸残基，并且AB IIa中的Glu10和Arg60被AB IIc中的Gln10和His60取代。AB IIa与AB I的比较显示，在AB IIa的78个残基中有25个变异氨基酸；此外，AB IIa在AB I的C末端区域缺少4个氨基酸残基。
