Heat‐Induced Fragmentation of the Maize Waxy Protein During Protein Extraction from Starch Granules

Abstract

ABSTRACTThe starch granule of maize contains a characteristic set of tightly bound polypeptides. Granule‐associated polypeptides are typically extracted from starch granules by heating starch granule suspensions at 90–100°C in a detergent such as SDS. Solubilized proteins are recovered by centrifugation and analyzed by gel electrophoresis. Previously identified tightly bound granule intrinsic proteins consist of the 85‐kDa starch‐branching enzyme IIb, the 76‐kDa starch synthase I, and the 60‐kD waxy (Wx) protein, also known as granule‐bound starch synthase 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 study, we analyzed this group of granule‐associated proteins and found that 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 immunoreactive polypeptide between 47 and 32 kDa was a heat‐induced fragmentation product of the Wx protein. Conditions for the extraction of granule proteins were evaluated. Our results demonstrate that granule proteins are effectively released by mild extraction (10‐min incubation at 72°C). Relative to the Wx protein, starch synthase I and starch branching enzyme IIb were less susceptible to thermal fragmentation. These results demonstrate that the 85‐, 76‐, and 60‐kDa polypeptides are authentic granule‐intrinsic proteins, and that the majority of polypeptides between 47 and 32 kDa are artifacts of high‐temperature granule extraction procedures.