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Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.

Crofts N, Abe N, Oitome NF, Matsushima R, Hayashi M, Tetlow IJ, Emes MJ, Nakamura Y, Fujita N - J. Exp. Bot. (2015)

Bottom Line: This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species.Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes.These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Production, Akita Prefectural University, 241-438 Kaidobata-Nishi, Shimoshinjo-Nakano, Akita city, Akita 010-0195, Japan.

No MeSH data available.


Related in: MedlinePlus

Expression and solubility of starch biosynthetic enzymes in rice endosperm, and confirmation of antibody specificity. Total (T), soluble (S), and insoluble, starch granule-associated (P) proteins were fractionated from rice developing endosperm and separated by SDS–PAGE. The gels were stained with Coomassie brilliant blue (CBB) or blotted onto membranes for western blotting using the antibodies indicated.
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Figure 1: Expression and solubility of starch biosynthetic enzymes in rice endosperm, and confirmation of antibody specificity. Total (T), soluble (S), and insoluble, starch granule-associated (P) proteins were fractionated from rice developing endosperm and separated by SDS–PAGE. The gels were stained with Coomassie brilliant blue (CBB) or blotted onto membranes for western blotting using the antibodies indicated.

Mentions: The expression and solubility of starch biosynthetic enzymes from rice developing endosperm (10–12 DAF) were analysed by western blotting (Fig. 1). Proteins were extracted using a denaturing buffer (see the Materials and methods) which also gelatinizes the starch to enable extraction of granule-bound proteins. Starch biosynthetic isozymes in developing rice endosperm (SSI, SSIIa, SSIIIa, SSIVb, GBSSI, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1; Hirose and Terao, 2004; Ohdan et al., 2005) were analysed by western blotting (Fig. 1). The antibodies used for western blots were highly specific and were visualized as single bands, except for the anti-SSIIIa antibody which recognized multiple bands. However, the ss3a mutants did not show any of the additional bands, suggesting that they represent truncated forms of SSIIIa (Supplementary Fig. S1 available at JXB online). Significant proportions of all the starch biosynthetic enzymes analysed here, except for GBSSI, were present in the soluble fraction. Currently it is unknown whether starch biosynthetic enzymes bound to the starch granule maintain their catalytic activities, except for GBSSI (Liu et al., 2009b). The solubility of the starch biosynthetic enzymes was consistent among the different extraction buffers used for the study (Supplementary Fig. S2) and were used for analyses of protein–protein interactions.


Amylopectin biosynthetic enzymes from developing rice seed form enzymatically active protein complexes.

Crofts N, Abe N, Oitome NF, Matsushima R, Hayashi M, Tetlow IJ, Emes MJ, Nakamura Y, Fujita N - J. Exp. Bot. (2015)

Expression and solubility of starch biosynthetic enzymes in rice endosperm, and confirmation of antibody specificity. Total (T), soluble (S), and insoluble, starch granule-associated (P) proteins were fractionated from rice developing endosperm and separated by SDS–PAGE. The gels were stained with Coomassie brilliant blue (CBB) or blotted onto membranes for western blotting using the antibodies indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4507757&req=5

Figure 1: Expression and solubility of starch biosynthetic enzymes in rice endosperm, and confirmation of antibody specificity. Total (T), soluble (S), and insoluble, starch granule-associated (P) proteins were fractionated from rice developing endosperm and separated by SDS–PAGE. The gels were stained with Coomassie brilliant blue (CBB) or blotted onto membranes for western blotting using the antibodies indicated.
Mentions: The expression and solubility of starch biosynthetic enzymes from rice developing endosperm (10–12 DAF) were analysed by western blotting (Fig. 1). Proteins were extracted using a denaturing buffer (see the Materials and methods) which also gelatinizes the starch to enable extraction of granule-bound proteins. Starch biosynthetic isozymes in developing rice endosperm (SSI, SSIIa, SSIIIa, SSIVb, GBSSI, BEI, BEIIa, BEIIb, ISA1, PUL, and Pho1; Hirose and Terao, 2004; Ohdan et al., 2005) were analysed by western blotting (Fig. 1). The antibodies used for western blots were highly specific and were visualized as single bands, except for the anti-SSIIIa antibody which recognized multiple bands. However, the ss3a mutants did not show any of the additional bands, suggesting that they represent truncated forms of SSIIIa (Supplementary Fig. S1 available at JXB online). Significant proportions of all the starch biosynthetic enzymes analysed here, except for GBSSI, were present in the soluble fraction. Currently it is unknown whether starch biosynthetic enzymes bound to the starch granule maintain their catalytic activities, except for GBSSI (Liu et al., 2009b). The solubility of the starch biosynthetic enzymes was consistent among the different extraction buffers used for the study (Supplementary Fig. S2) and were used for analyses of protein–protein interactions.

Bottom Line: This study investigated whether protein-protein interactions are also found in rice endosperm, as well as exploring differences between species.Blue-native-PAGE zymogram analyses confirmed the glucan-synthesizing activity of protein complexes.These results suggest that some rice starch biosynthetic isozymes are physically associated with each other and form active protein complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Production, Akita Prefectural University, 241-438 Kaidobata-Nishi, Shimoshinjo-Nakano, Akita city, Akita 010-0195, Japan.

No MeSH data available.


Related in: MedlinePlus