Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Abstract

Soluble starch synthase (SSS) I was purified 361‐fold from hand‐dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min−1 mg−1. A key to this purification protocol was the introduction of a size‐exclusion chromatography step, a size‐based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl‐Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.