HEAT-INDUCED FRAGMENTATION OF THE MAIZE WAXY PROTEIN DURING PROTEIN EXTRACTION FROM STARCH GRANULES

The starch granule of maize contains a characteristic set of tightly b ound polypeptides, Granule-associated polypeptides are typically extra cted from starch granules by heating starch granule suspensions at 9-1 00 degrees C in a detergent such as SDS. Solubilized proteins are reco vered by centrifugation and analyzed by gel electrophoresis. Previousl y identified tightly bound granule intrinsic proteins consist of the 8 5-kDa starch-branching enzyme IIb, the 76-kDa starch synthase I, and t he 60-kD waxy (Wx) protein, also known as granule-bound starch synthas e I. However, SDS extracts from starch granules of maize also contain a cluster of proteins ranging in mass between 47 and 32 kDa In this st udy, we analyzed this group of granule-associated proteins and found t hat each was recognized by the Wx antibody. A 15 amino acid N-terminal sequence from the 47-kDa polypeptide was identical to the predicted N -terminus of the Wx protein. Further analysis revealed that each immun oreactive polypeptide between 47 and 32 kDa was a heat-induced fragmen tation product of the Wx protein. Conditions for the extraction of gra nule proteins were evaluated. Our results demonstrate that granule pro teins are effectively released by mild extraction (10-min incubation a t 72 degrees C). Relative to the Wx protein, starch synthase I and sta rch branching enzyme IIb were less susceptible to thermal fragmentatio n. These results demonstrate that the 85-, 76-, and 60-kDa polypeptide s are authentic granule-intrinsic proteins, and that the majority of p olypeptides between 47 and 32 kDa are artifacts of high-temperature gr anule extraction procedures.