The goal of this research is to understand the biochemical lesion of the sugary1 mutation in maize. The sugary1 gene is a determinant of starch structure. Homozygous su1-mutant endosperms accumulate a highly-branched water-soluble polysaccharide, phytoglycogen, at the expense of amylopectin. These suggest that both branched polysaccharides share a common biosynthetic pathway, and that SU1 participates in the biosynthesis of kernel starch. Specific antibodies were used to demonstrate that SU1 is a 79-kD protein that accumulates in endosperm during starch biosynthesis. Recombinant near full-length SU1 (SU1r) was expressed in E. coli and purified to apparent homogeneity. Determination of substrate specificity of SU1r identified it as an isoamylase-type debranching enzyme (DBE). Previous studies had shown that su1 endosperms are deficient in a different DBE, a pullulanase. Immunoblot analysis with anti-SU1 and anti-pullulanase revealed that both proteins are missing from su1 kernels;A maize pullulanase cDNA, Zpu1, was cloned based on its homology with a rice pullulanase cDNA. The zpu1 gene was found as a single copy in maize genome which mapped to chromosome 2. Comparison of zPU1 and other DBEs identified sequence blocks conserved in both pullulanases and isoarnylases, and some class-specific sequence blocks. Zpu1 mRNA was abundant in endosperm throughout starch biosynthesis, but was not detected in leaf or root. Both pullulanase- and isoamylase-type DBE activities were purified from developing maize kernels. These DBE activities were identified as zPU1 and SU1, respectively. zPU1 was greatly reduced in su1-mutant kernels, indicating this is the affected pullulanase. Zpu1 transcripts were of equivalent size and abundance in nonmutant and sul-mutant kernels, indicating that coordination regulation of the DBEs occur post-transcriptionally;SU1 was also purified to apparent homogeneity from maize endosperm. The mature N terminus was identified demonstrating that the primary translation product contains a 49 ainino acid N terminal extension that is proteolytically cleaved to produce mature SU1. The molecular weight of native SU1 was shown to be greater than 670,000 Da. SU1 was shown to bind to itself using the yeast two-hybrid system. Thus mature SU1 is a homomultimeric enzyme.