Identification of the maize amyloplast stromal 112-kD protein as a plastidic starch phosphorylase

Amyloplast is the site of starch synthesis in the storage tissue of maize ( Zea mays). The amyloplast stroma contains an enriched group of proteins whe n compared with the whole endosperm. Proteins with molecular masses of 76 a nd 85 kD have been identified as starch synthase I and starch branching enz yme Ire, respectively. A 112-kD protein was isolated from the stromal fract ion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjec ted to tryptic digestion and amino acid sequence analysis. Three peptide se quences showed high identity to plastidic forms of starch phosphorylase (SP ) from sweet potato, potato, and spinach. SP activity was identified in the amyloplast stromal fraction and was enriched 4-fold when compared with the activity in the whole endosperm fraction. Native and sodium dodecyl sulfat e-polyacrylamide gel electrophoresis analyses showed that SP activity was a ssociated with the amyloplast stromal 112-kD protein. In addition, antibodi es raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole en dosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amy loplast stromal 112-kD SP preferred amylopectin over,glycogen as a substrat e in the synthetic reaction. The maize shrunken-4 mutant had reduced SP act ivity due to a decrease of the amyloplast stromal 112-kD enzyme.