ASSOCIATION OF A 76-KDA POLYPEPTIDE WITH SOLUBLE STARCH SYNTHASE I ACTIVITY IN MAIZE (CV B73) ENDOSPERM

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissec ted endosperm tissue of inbred maize (Zea mays, cv. B73) to specific a ctivities ranging between 5 and 9 mu mol min(-1) mg(-1). A key to this purification protocol was the introduction of a size-exclusion chroma tography step, a size-based fractionation which provided abundant leve ls of desalted SSS forms I and II. The native molecular masses calcula ted for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SS SI 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 gener ated against a 76 kDa polypeptide extracted from starch granules. Thes e antibodies, which were monospecific for the soluble 76 kDa polypepti de, neutralized greater than 90% of SSSI activity, and precipitated th e 76 kDa protein. These results establish the 76 kDa protein as an SSS I in the B73 line of inbred maize. An immunologically similar 76 kDa p rotein also appears to be tightly associated with the starch granule.