Abstract:

The factors affecting the binding characteristics of GBSS with starch granule were studied using a wheat (Triticum aestivum) cultivar Chinese Spring with different temperature treatments. After sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis, we got GBSS concentration by coomassie brilliant blue G-250(CBB G-250) method. The results showed that the temperature affected the binding of GBSS and starch granule. In 50–80℃, the concentration of GBSS unbound from starch increased with the temperature rising, which the maximal concentration was 11.361 μg mL^-1 at 80℃, while that was reduced when the temperature was 85–95℃. Furthermore, Mg²⁺ could also affect the quantity of GBSS unbound from starch. When Mg2+ concentration was lower than 1.75 mmol L^-1, the concentration of GBSS unbound increased. The lower Mg²⁺ concentration, the higher the concentration of GBSS unbound. However, when Mg2+ concentration was higher than 2.5 mmol L^-1, it could restrain GBSS unbinding. These results showed that GBSS bound starch granule by non-covalent bonds. The best GBSS extracting conditions were 55 mmol L^-1 Tris-HCl (pH 6.8), 0.75 mmol L^-1 MgCl₂, 2.3% SDS, 5% 2-ME and 10% glycerol, 15 min in boiling water, or 55 mmol L^-1 Tris-HCl (pH 6.8), 0.75 mmol L^-1 MgCl₂, 2.3% SDS, 5% 2-ME and 10% glycerol, 80℃ 30 min. The results are helpful to investigate 3-D structure of biological activated GBSS and mechanism of GBSS binding with starch granule.

Key words: Wheat, Granule-Bound Starch Synthase (GBSS), Binding character, Temperature, Mg²⁺ concentration