Purification and some properties of a subtilisin inhibitor from adzuki beans.

Abstract

A subtilisin inhibitor was isolated from adzuki beans (Phaseolus angularis) by chromatography on CM-cellulose, Sephadex G-75, DEAE-cellulose, and SP-Sephadex C-25. The final preparation was confirmed to be homogeneous on polyacrylamide gel electrophoreses, and its pi value was 3.7. The preparation was a powerful inhibitor of microbial serine-proteinases but its activity was destroyed by trypsin and chymotrypsin. Dissociation constant of the complex of the inhibitor with subtilisin was 0.16 nM. The inhibitor was heat-stable over the pH range examined in spite of a lack of intramolecular disulfide bonds: little or no subtilisin-inhibitory activity was lost at 80°C for lOmin, though heating to 100°C at.pH 12 caused a decrease of about 50% in the activity. The inhibitor molecule consisted of 97 amino acid residues, containing relatively large amounts of glutamic acid and valine residues and no half-cystine residues, unlike adzuki-bean proteinase inhibitors I and II. The molecular weight of the inhibitor was determined to be 12,300 by gelfiltration and calculated to be approximately 1 1,000 on the basis of the amino acid composition.

Protease Inhibitors in Plants: Genes for Improving Defenses Against Insects and Pathogens

“…Other inhibitors of subtilisin were sub sequently purified from cereals and legumes. Subtilisin inhibitors are found in Hordeum vulgare (barley seeds) (17,191), Vigna unguiculata (cowpea) (180), Vicia faba (broad beans) (170), Phaseolus vulgaris (black beans) (160), Phaseolus angularis (adzuki bean) (192), Vigna radiata (mung bean) (89), Cicer arietinum (chick pea) (176) and Canavalia ensiformis (jack beans) (161). These inhibitors from legumes and cereals were first characterized by their high specificities toward microbial proteinases and their relative in effectiveness against serine proteinases from animals.…”

Proteinase inhibitors and their function in plants: A review

“…Another group of related proteins from leguminous plants comprises subtilisin inhibitors from common garden bean ( Phaseolus vulgaris L.) [128], adzuki bean ( P. angularis L.) [129], broad bean ( Vicia faba L.) [22], and jack bean ( Canavalia lineata L.) [130]. In addition to subtilisin, inhibitors of leguminous plants act on other serine proteinases of microorganisms, but not trypsin.…”

“…Although the inhibitors of this family either lack disulfides or have only one such bond per molecule, they are highly pH-stable and resistant to temperature fluctuations [121,129,136]. Moreover, it was shown that reduction of the single disulfide bond in the molecule of potato chymotrypsin inhibitor did not affect its conformation or activity [138].…”

“…As indicated above, many plants contain, in addition to trypsin or chymotrypsin inhibitors, proteins that act preferentially on proteinases of microbial origin [21,22,[126][127][128][129]. One of the most specific inhibitors of microbial serine proteinases was isolated from a common garden bean of the cultivar Tweed wonder [340].…”

Neutrophil elastase inhibitor purification strategy from cowpea seeds

“…Adzuki Subtilisin Inhibitor (ASI), an inhibitor purified from Vigna angularis , can inhibit subtilisin with a Ki of 0.16 nM. The 10.8 kDa molecule was identified as being from the PotatoI family through amino acid sequence analysis, and was purified by CM-cellulose, Sephadex G-75, DEAE-Cellulose and Sephadex C-25 [38, 39]. Plant inhibitors do not inhibit only serine proteases, in Vigna radiate an inhibitor of aspartic protease (VrAP) of 16.6 kDa with Ki in nM range was characterized.…”