Proteinase Inhibitor from Vicia angustifolia L. var. segetalis Koch
Isolation, Characterization, and Chemical Modification
1978 年 83 巻 6 号 p. 1737-1748
詳細
1978 年 83 巻 6 号 p. 1737-1748
A proteinase inhibitor which inhibits trypsin [EC 3. 4. 21. 4] and α-chymotrypsin [EC 3. 4. 21. 1] was isolated from the seeds of Vicia angustifolia L. var. segetalis Koch and purified by gel filtration on Sephadex G-100 followed by ion-exchange chromatography on DEAE-Sephadex A-25. The inhibitor has a molecular weight of about 13, 000 when determined by molecularsieve chromatography on Sephadex G-100 and 12, 600 from the amino acid composition, which revealed high contents of half-cystine, aspartic acid, serine, and glutamic acid residues. The absence of free sulfhydryl groups, tryptophan, and carbohydrate was also observed. The amino- and carboxyl-terminal residues of the inhibitor were determined to be glycine and asparagine, respectively, suggesting that this inhibitor is composed of a single polypeptide chain. The inhibitor has an isoelectric point at pH 7.6 and is considerably stable in acidic and neutral pH regions, however, it loses inhibitory activities at alkaline pH.
Stoichiometry of the reactions between the inhibitor and the enzymes showed that this inhibitor reacted with trypsin in a 1:1 and with chymotrypsin in a 1:2 molar ratio.
Maleylation of the inhibitor revealed that even when all the 9 amino groups, i.e. one α-amino and 8 ε-amino groups, were modified, the inhibitor retained its full antitryptic activity. On the other hand, on 1, 2-cyclohexanedione treatment, the inhibitor lost almost all the antitryptic activity and also 50% of the antichymotryptic activity. Modifications of all the three tyrosyl residues in the inhibitor decreased the antichymotryptic activity by 50% but had no effect on the antitryptic activity. The results of these chemical modifications seem to support that this inhibitor has one reactive site for trypsin and two for α-chymotrypsin.
