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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

BN-PAGE activity staining shows the glucan synthesis activities of starch biosynthetic enzyme complexes from developing rice endosperm. (A) CBB-stained BN-PAGE gel showing separation of protein complexes. (B) Synthesis of glucan by endogenous BE–Pho1 interaction. The BN-PAGE gel was incubated with 50mM G1P, and the generated glucans were visualized by iodine staining. (C) Stimulation of glucan synthesis by exogenous phosphorylase a (Pho a). The BN-PAGE gel was incubated with 50mM G1P and rabbit Pho a, and stained with iodine. Black arrowheads indicate glucan synthesis activity by interaction of endogenous BEs and Pho1. The white arrowhead indicates glucan synthesis arising from co-migration of BEs and Pho1 due to their similar monomeric sizes. Arrows and the bracket indicate the stimulation of glucan synthesis by addition of exogenous Pho a. Single asterisks indicate residual CBB from the BN-PAGE running buffer (not glucans stained by iodine). The activity band indicated with double asterisks corresponds to SSI, BEI, BEIIb, and Pho1as indicated in Fig. 5
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Figure 6: BN-PAGE activity staining shows the glucan synthesis activities of starch biosynthetic enzyme complexes from developing rice endosperm. (A) CBB-stained BN-PAGE gel showing separation of protein complexes. (B) Synthesis of glucan by endogenous BE–Pho1 interaction. The BN-PAGE gel was incubated with 50mM G1P, and the generated glucans were visualized by iodine staining. (C) Stimulation of glucan synthesis by exogenous phosphorylase a (Pho a). The BN-PAGE gel was incubated with 50mM G1P and rabbit Pho a, and stained with iodine. Black arrowheads indicate glucan synthesis activity by interaction of endogenous BEs and Pho1. The white arrowhead indicates glucan synthesis arising from co-migration of BEs and Pho1 due to their similar monomeric sizes. Arrows and the bracket indicate the stimulation of glucan synthesis by addition of exogenous Pho a. Single asterisks indicate residual CBB from the BN-PAGE running buffer (not glucans stained by iodine). The activity band indicated with double asterisks corresponds to SSI, BEI, BEIIb, and Pho1as indicated in Fig. 5

Mentions: Incubation of the BN-PAGE gel (Fig. 6A; CBB stained) with G1P followed by iodine staining demonstrated α-glucan synthesis in a wide molecular weight range of protein complexes, at ~100, 200, 440, 500, and 1000kDa (Fig. 6B). The 100kDa glucan band was likely to be generated by co-migration of monomeric Pho1 and BEs (BEIIa and BEIIb) as observed by western blotting of the BN-PAGE gel (Fig. 5; Supplementary Fig. S4 at JXB online). Addition of exogenous Pho a to the G1P-containing reaction mixture led to a significant increase in amounts of generated α-glucans at ~400–600kDa and ~1000kDa. A very discrete brown band at ~670kDa and the two, nearby, lower bands became apparent (Fig. 6C), indicating the presence of BE activities which are able to interact with the exogenous Pho a.


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)

BN-PAGE activity staining shows the glucan synthesis activities of starch biosynthetic enzyme complexes from developing rice endosperm. (A) CBB-stained BN-PAGE gel showing separation of protein complexes. (B) Synthesis of glucan by endogenous BE–Pho1 interaction. The BN-PAGE gel was incubated with 50mM G1P, and the generated glucans were visualized by iodine staining. (C) Stimulation of glucan synthesis by exogenous phosphorylase a (Pho a). The BN-PAGE gel was incubated with 50mM G1P and rabbit Pho a, and stained with iodine. Black arrowheads indicate glucan synthesis activity by interaction of endogenous BEs and Pho1. The white arrowhead indicates glucan synthesis arising from co-migration of BEs and Pho1 due to their similar monomeric sizes. Arrows and the bracket indicate the stimulation of glucan synthesis by addition of exogenous Pho a. Single asterisks indicate residual CBB from the BN-PAGE running buffer (not glucans stained by iodine). The activity band indicated with double asterisks corresponds to SSI, BEI, BEIIb, and Pho1as indicated in Fig. 5
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4507757&req=5

Figure 6: BN-PAGE activity staining shows the glucan synthesis activities of starch biosynthetic enzyme complexes from developing rice endosperm. (A) CBB-stained BN-PAGE gel showing separation of protein complexes. (B) Synthesis of glucan by endogenous BE–Pho1 interaction. The BN-PAGE gel was incubated with 50mM G1P, and the generated glucans were visualized by iodine staining. (C) Stimulation of glucan synthesis by exogenous phosphorylase a (Pho a). The BN-PAGE gel was incubated with 50mM G1P and rabbit Pho a, and stained with iodine. Black arrowheads indicate glucan synthesis activity by interaction of endogenous BEs and Pho1. The white arrowhead indicates glucan synthesis arising from co-migration of BEs and Pho1 due to their similar monomeric sizes. Arrows and the bracket indicate the stimulation of glucan synthesis by addition of exogenous Pho a. Single asterisks indicate residual CBB from the BN-PAGE running buffer (not glucans stained by iodine). The activity band indicated with double asterisks corresponds to SSI, BEI, BEIIb, and Pho1as indicated in Fig. 5
Mentions: Incubation of the BN-PAGE gel (Fig. 6A; CBB stained) with G1P followed by iodine staining demonstrated α-glucan synthesis in a wide molecular weight range of protein complexes, at ~100, 200, 440, 500, and 1000kDa (Fig. 6B). The 100kDa glucan band was likely to be generated by co-migration of monomeric Pho1 and BEs (BEIIa and BEIIb) as observed by western blotting of the BN-PAGE gel (Fig. 5; Supplementary Fig. S4 at JXB online). Addition of exogenous Pho a to the G1P-containing reaction mixture led to a significant increase in amounts of generated α-glucans at ~400–600kDa and ~1000kDa. A very discrete brown band at ~670kDa and the two, nearby, lower bands became apparent (Fig. 6C), indicating the presence of BE activities which are able to interact with the exogenous Pho a.

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