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

Abstract

Amyloplast is the site of starch synthesis in the storage tissue of maize (Zea mays). The amyloplast stroma contains an enriched group of proteins when compared with the whole endosperm. Proteins with molecular masses of 76 and 85 kD have been identified as starch synthase I and starch branching enzyme IIb, respectively. A 112-kD protein was isolated from the stromal fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and subjected to tryptic digestion and amino acid sequence analysis. Three peptide sequences 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 sulfate-polyacrylamide gel electrophoresis analyses showed that SP activity was associated with the amyloplast stromal 112-kD protein. In addition, antibodies raised against the potato plastidic SP recognized the amyloplast stromal 112-kD protein. The amyloplast stromal 112-kD SP was expressed in whole endosperm isolated from maize harvested 9 to 24 d after pollination. Results of affinity electrophoresis and enzyme kinetic analyses showed that the amyloplast stromal 112-kD SP preferred amylopectin over glycogen as a substrate in the synthetic reaction. The maize shrunken-4 mutant had reduced SP activity due to a decrease of the amyloplast stromal 112-kD enzyme.

Figure 1

SDS-PAGE analysis of the endosperm and amyloplast stromal fraction and immunoblot analysis of endosperm, amyloplast stromal, and granule fractions. A, Samples (30 μg) of the endosperm (En) and amyloplast stromal fraction (Am) were subjected to SDS-PAGE followed by Coomassie Blue staining. The molecular mass standards from top to bottom are myosin (200 kD), β-galactosidase (116.2 kD), phosphorylase b (97.4 kD), bovine serum albumin (66.2 kD), ovalbumin (45 kD), and carbonic anhydrase (31 kD). B, A sample (0.1 μg) of the isolated 112-kD protein and samples (30 μg) of the endosperm, amyloplast stromal, and granule fractions were subjected to immunoblot analysis using anti-SP antibodies. C, Samples (30 μg) of the endosperm, amyloplast stromal, and granule fractions were subjected to immunoblot analysis using anti-starch synthase I antibodies. A portion of the immunoblots is shown in B and C. The position of the 112-kD protein is indicated in A and B, and the position of starch synthase I (SSI) is indicated in C. The data shown in each A through C is representative of two independent experiments.

Figure 2

Amino acid sequences of the amyloplast stromal 112-kD protein and alignment with amino acid sequences of plastidic and cytosolic forms of potato SP. The isolated 112-kD protein was subjected to SDS-PAGE. The protein was then transferred to polyvinylidene difluoride paper and subjected to N-terminal amino acid sequence analysis. Another sample of the protein was digested with trypsin, three peptides were isolated, and subjected to amino acid sequence analysis. Sequences of the N terminus and from three internal peptides of the 112-kD protein were aligned with published sequences of plastidic SP (Nakano and Fukui, 1986; Nakano et al., 1989) and cytosolic SP (Mori et al., 1991) from potato tuber. The numbers in the figure represent the residue numbers that begin with the indicated published sequences.

Figure 3

Dependence of the relative mobility of the 112-kD stromal SP on the concentration of amylopectin and glycogen upon affinity electrophoresis. Samples (30 μg) of the amyloplast stromal fraction were subjected to native PAGE in the presence of the indicated concentrations of amylopectin (A) and glycogen (B). Following electrophoresis, SP activity was measured by iodine staining. The relative mobility was calculated by dividing the migration of the activity band by the migration of the dye front. The inset of A and B contains a portion of the native gels showing the relative mobility of SP. The data shown in A and B is representative of two independent experiments.

Figure 4

Dependence of SP activity on the concentration of amylopectin and glycogen. SP activity was measured as a function of the indicated concentrations of amylopectin (A) and glycogen (B). The concentration of Glc-1-P was maintained at 20 mm. The curves drawn were the result of the analysis of the data according to the Michaelis-Menten equation. The insets shown in A and B are double reciprocal plots of the data. The lines drawn in the insets are the result of a least-squares analysis of the data. The data shown in A and B is representative of two independent experiments carried out in duplicate.

Figure 5

Levels of the 112-kD stromal SP in the shrunken-4 mutant. A, Samples (30 μg) of the endosperm fraction from wild-type (WT) and the shrunken-4 (sh-4) mutant were subjected to native PAGE in the presence of 24 μm glycogen. Following electrophoresis, SP activity was measured by iodine staining. B, The isolated 112-kD protein (0.1 μg) and samples (60 μg) of the endosperm fraction from wild-type (WT) and the shrunken-4 (sh-4) mutant were subjected to SDS-PAGE followed by Coomassie Blue staining. C, The isolated 112-kD protein (0.1 μg) and samples (30 μg) of the endosperm fraction from wild-type and the shrunken-4 mutant were subjected to immunoblot analysis using anti-SP antibodies. A portion of the polyacrylamide gels (A and B) and the immunoblot (C) is shown, and the position of the 112-kD stromal SP is indicated in the figure. The data shown in A through C is representative of two independent experiments.