A high-molecular-weight a-amylase inhibitor has been isolated from mature tubers of Dioscorea a h a by extraction with 0 . 0 2~ acetate buffer (pH 5.0), negative absorption on CM-cellulose and ultracentrifuging. The inhibitor was fairly heat stable and was active against human salivary, human pancreatic and pig pancreatic amylases. The inhibitor had no action on Bacillus sitbtilis and Aspergillus oryzae amylases. Trypsin and a-chymotrypsin inactivated the inhibitor. Pre-incubation of the inhibitor with starch or concanavalin A resulted in complete abolition of its activity. Chemical modification of the amino groups with trinitrobenzenc sulphonic acid led to loss of activity. The inhibitor was found to be a glycoprotein with 64% carbohydrate. The monosaccharide units present were glucose, mannose and galactose in a molar ratio of 5.5:3.8: 1 .
I . I. TihersYam (tubers of D. ulutn) was procured from commercial sources locally.
Chemiculs and erizymes
Two n-amylase inhibitors 1-1 and 1-2, were purified to homogeneity from Colocasia mtiquorunz tubers by extraction with 0.02M phosphate buffer pH 7.6, treatment with diethyl amino ethyl-cellulose, ammonium sulphate fractionation, chromatography on Sephadex G-50 and chromatography on phenyl sepharose CL-4B. Both the inhibitors were basic proteins. 1-1 alone contained carbohydrates to the extent of 5.4%. The molecular weights of 1-1 and 1-2 were found to be about 14 300 and 12 500, respectively, by sodium dodecyl sulphate-polyacrylaniide gel electrophoresis. The inhibitors inactivated human salivary, human pancreatic and hog pancreatic amylases but they had no action on Bacillus subtilis and Aspergillirs oryzae amylases and resisted attack by pepsin, trypsin, chymotrypsin and pronase. Chemical modification of amino groups and guanidino groups of 1-1 and 1-2 resulted in loss of inhibitory activities. Formation of enzyme inhibitor complexes between hog pancreatic amylase, 1-1 and 1-2 were demonstrated by gel chromatography on Sephadex G-100. Total amylase inhibitory activity in Colocasia tubers decreased gradually during plant growth.
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