Purification and partial characterization of white kidney bean (Phaseolus vulgaris) alpha-amylase inhibitors from two experimental cultivars
1993
Ho, M.F. | Whitaker, J.R.
alpha-Amylase inhibitors WKB 858A and WKB 858B were purified to homogeneity from different cultivars of white kidney beans by extraction from the ground beans and by sequential heat treatment, ethanol fractionation, DEAE-cellulose chromatography, Sephadex G-75 gel chromatography and CM-cellulose chromatography. The inhibitors were homogeneous by 7.5% polyacrylamide gel electrophoresis; no isoinhibitors were found Inhibitors WKB 858A and WKB 858B had isoelectric points of 5.0 and 4.65, respectively, and molecular weights of 42, 000 and 20, 000, respectively, by FPLC Superose 12 gel filtration chromatography. Inhibitor WKB 858A had molecular weights of 40, 000 and 38, 000 by Sephadex G-75 gelfiltration chromatography and by native gel electrophoresis, respectively. Inhibitor WKB 858A contained 11.0% carbohydrate, N-linked to asparagine residues, with a composition of 1 fucose, 1 xylose, 4 galactose, 8 N-acetylglycosamine and 13 mannose residues per mol of inhibitor. Amino acid analysis of Inhibitor WKB 858A gave a high content of Asx, Glx, Ser, Thr and Val (combined total of 60 % molar ratio) and low content of sulfur amino acids (0.8% molar ratio of Met and no 1/2 cystine). No -SH groups were found. The amino acid composition was similar to that of eight other alpha-amylase inhibitors from beans. Inhibitor WKB 858A formed 1:1 stoichiometric complex with porcine pancreatic alpha-amylase with a Ki of 1.0 X 10-11 M at pH 5.4 and 3OC; it had no trypsin inhibitory activity. At pH 6.90 and 30C, the rate of complex formation between Inhibitor WKB 858A and porcine pancreatic alpha-amylase was 2.76 times faster at 1.385 vs 0.035 ionic strength (with Na2SO4), indicating hydrophobic bonds are most important in complex formation.
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