Simplified electrophoretic assay for human salivary alpha-amylase inhibitor detection in cereal seed flours. 2007

Debora Fontanini, and Antonella Capocchi, and Franco Saviozzi, and Luciano Galleschi
Department of Biology, University of Pisa, Via L. Ghini 5, 56126 Pisa, Italy. dfontanini@biologia.unipi.it

Alpha-amylase inhibitors are antinutritional proteins largely found in cereal seeds. An in-gel assay was developed that allowed the rapid screening of these compounds in complex seed extracts. The assay was based on the electrophoretic separation of the extract proteins on starch-containing gels, followed by the detection of alpha-amylase-inhibiting proteins after incubation of the gel in an alpha-amylase solution; inhibitors were revealed by a staining method based on iodine binding to nondigested starch. The one-dimensional method can be useful to test inhibitory activity of purified proteins or to assay fractions recovered during a purification procedure. A two-dimensional (IEF x PAGE) non-denaturing system with second-dimension separation on starch-PAGE was also developed; the technique allowed the screening of complex protein mixtures for multiple inhibitory proteins. The newly developed assay method was used to test the presence of inhibitory activity in a crude extract from wheat flour, and it was validated by comparing in-gel and in-solution assays of commercially available alpha-amylase inhibitors.

UI MeSH Term Description Entries
D004591 Electrophoresis, Polyacrylamide Gel Electrophoresis in which a polyacrylamide gel is used as the diffusion medium. Polyacrylamide Gel Electrophoresis,SDS-PAGE,Sodium Dodecyl Sulfate-PAGE,Gel Electrophoresis, Polyacrylamide,SDS PAGE,Sodium Dodecyl Sulfate PAGE,Sodium Dodecyl Sulfate-PAGEs
D004791 Enzyme Inhibitors Compounds or agents that combine with an enzyme in such a manner as to prevent the normal substrate-enzyme combination and the catalytic reaction. Enzyme Inhibitor,Inhibitor, Enzyme,Inhibitors, Enzyme
D005433 Flour A powdery meal obtained from ground up starchy foodstuff, such as seeds of WHEAT; RICE and other EDIBLE GRAIN; LEGUMES; and NUTS; or ground up PLANT TUBERS. Almond Flour,Almond Meal,Corn Flour,Corn Meal,Grain Flour,Grain Meal,Nut Flour,Potato Flour,Rice Flour,Rice Meal,Soybean Flour,Soybean Meal,Wheat Flour,Wheat Meal,Semolina Flour,Almond Flours,Almond Meals,Corn Flours,Corn Meals,Flour, Almond,Flour, Corn,Flour, Grain,Flour, Nut,Flour, Potato,Flour, Rice,Flour, Semolina,Flour, Soybean,Flour, Wheat,Flours,Flours, Almond,Flours, Corn,Flours, Grain,Flours, Nut,Flours, Potato,Flours, Rice,Flours, Semolina,Flours, Soybean,Flours, Wheat,Grain Flours,Grain Meals,Meal, Almond,Meal, Corn,Meal, Grain,Meal, Rice,Meal, Soybean,Meal, Wheat,Meals, Almond,Meals, Corn,Meals, Grain,Meals, Rice,Meals, Soybean,Meals, Wheat,Nut Flours,Potato Flours,Rice Flours,Rice Meals,Semolina Flours,Soybean Flours,Soybean Meals,Wheat Flours,Wheat Meals
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000516 alpha-Amylases Enzymes that catalyze the endohydrolysis of 1,4-alpha-glycosidic linkages in STARCH; GLYCOGEN; and related POLYSACCHARIDES and OLIGOSACCHARIDES containing 3 or more 1,4-alpha-linked D-glucose units. Taka-Amylase A,alpha-Amylase,Alpha-Amylase Bayer,Maxilase,Mégamylase,alpha-1,4-D-Glucanglucanohydrolase,Alpha Amylase Bayer,AlphaAmylase Bayer,Taka Amylase A,TakaAmylase A,alpha 1,4 D Glucanglucanohydrolase,alpha Amylase,alpha Amylases
D012463 Saliva The clear, viscous fluid secreted by the SALIVARY GLANDS and mucous glands of the mouth. It contains MUCINS, water, organic salts, and ptylin. Salivas
D012639 Seeds The encapsulated embryos of flowering plants. They are used as is or for animal feed because of the high content of concentrated nutrients like starches, proteins, and fats. Rapeseed, cottonseed, and sunflower seed are also produced for the oils (fats) they yield. Diaspores,Elaiosomes,Embryos, Plant,Plant Embryos,Plant Zygotes,Zygotes, Plant,Diaspore,Elaiosome,Embryo, Plant,Plant Embryo,Plant Zygote,Seed,Zygote, Plant
D014908 Triticum A plant genus of the family POACEAE that is the source of EDIBLE GRAIN. A hybrid with rye (SECALE CEREALE) is called TRITICALE. The seed is ground into FLOUR and used to make BREAD, and is the source of WHEAT GERM AGGLUTININS. Wheat,Durum Wheat,Triticum aestivum,Triticum durum,Triticum spelta,Triticum turgidum,Triticum turgidum subsp. durum,Triticum vulgare,Durum Wheats,Wheat, Durum

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