Purification and Some Physical and Chemical Properties of Red Kidney Bean (Phaseolus Vulgaris) ?-Amylase Inhibitor

Abstract

Red kidney bean contains more amylase inhibitor than do California white bean and cowpea while garbanzo bean and Westan and Westley lima beans do not contain inhibitor. Red kidney bean amylase inhibitor was purified to homogeneity by selective heat treatment (60°C) of a water extract at pH 4. 0, fractionation with ethanol and successive chromatography on DEAE‐ and CM‐cellulose chromatography. The inhibitor has an apparent molecular weight of 49,000 by Sephadex gel filtration and contains 8. 6% carbohydrate probably covalently linked via an amide linkage to asparagine. The inhibitor probably contains four subunits perhaps of three different types. The inhibitor is high in aspartic acid, glutamic acid, serine, threonine and valine, low in cysteine/cystine and does not contain proline. Stable 1:1 complex formation between inhibitor and porcine pancreatic α‐amylase was demonstrated by gel filtration on Sephadex G‐100. The inhibitor has activity against porcine pancreatic α‐amylase, human salivary α‐amylase, and Tenebrio molitor (yellow corn meal worm) larval midgut α‐amylase but is inactive against Bacillus subtilis α‐amylase, Aspergillus oryzae α‐amylase, barley α‐amylase and red kidney bean α‐amylase.

Characterization of α-Amylase-Inhibitor, a Lectin-Like Protein in the Seeds of Phaseolus vulgaris

“…Polypeptides b and c had as their dominant sequence amino acids 24 to 34 of the derived sequence of the LLP gene, and the other sequence as a contaminant. These results imply that the five polypeptides, a to e, may be glycoforms of two different polypeptides, since it is known that aAI is a glycoprotein (24,25).…”

“…Midmature (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) d after pollination) cotyledons of seeds of the common bean (Phaseolus vulgaris cv Greensleeves) were homogenized in an ice-cold mortar with 3 mL of 10 mM f3-mercaptoethanol per g of fresh weight. The homogenate was centhftiged at 12,000g for 10 min and the supematant was buffered by adding 0.2 M succinate, 0.1 M CaCl2 (pH 3.8) (1 10 ,uL/mL) and was heated in a water bath at 70°C for 10 min.…”

“…9, lane 2 (19,24,25), postulating a trimer or tetramer structure on the basis of the mol wt of the subunits and of the native inhibitor as determined by gel filtration. However, some authors have pointed out that the mol wt of aAI could be lower than calculated due to the abnormal behavior of glycoproteins in gel filtration columns (25). The (5).…”

“…The aAI has been characterized in several varieties of beans (19,21,25). It is a glycoprotein (about 15% carbohydrate) and its native mol wt has been estimated to be 43 to 50 kD by gel filtration experiments (24,25).…”

“…It is a glycoprotein (about 15% carbohydrate) and its native mol wt has been estimated to be 43 to 50 kD by gel filtration experiments (24,25). The inhibitor is composed of subunits of Mr 15,000 to 18,000, and it has been proposed that it is either a trimer or a tetramer of identical polypeptides (24) or different polypeptides (19,25). The inhibitor binds to animal a-amylases at a pH optimum of 5.6, forming a stable 1:1 (molar ratio) complex (26,34).…”