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

¹Department of Food Science, New Jersey Agricultural Experiment Station, Cook College, Rutgers University, 65 Dudley Rd., New Brunswick NJ 08901-8520. Phone: 732/932-9611 ext. 220. Fax: 732/932-6776. ²Present address: 800 N. Lindbergh Blvd. St. Louis, MO 63198. ³Corresponding author. E-mail: wasserman@aesop.rutgers.edu

The 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.