Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits
Accumulation of β-conglycinin in soybean cotyledon through the formation of disulfide bonds between α'- and α-subunits
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通过在 α- 和 α- 亚基之间形成二硫键，β-伴大豆球蛋白在大豆子叶中积累
DOI：
10.1104/pp.111.189621
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发表时间：
2012
期刊：
影响因子：
7.4
通讯作者：
Ishimoto M
中科院分区：
文献类型：
--
作者：
Wadahama H;Iwasaki K;Matsusaki M;Nishizawa K;Ishimoto M
β-伴大豆球蛋白是主要的大豆 (Glycine max) 种子储存蛋白之一，在内质网中折叠并组装成三聚体，并积累到蛋白质储存液泡中。先前的实验使用了大豆β-伴大豆球蛋白，该大豆β-伴大豆球蛋白是通过含有巯基还原剂（例如2-巯基乙醇）的还原缓冲液提取的，该还原剂可还原蛋白质内的分子间和分子内二硫键。在这项研究中，在非还原条件下从未成熟种子或干豆的子叶中提取大豆蛋白，以防止硫醇基团的氧化和二硫键的还原或交换。我们发现，在氨基末端前肽加工之前，β-伴大豆球蛋白的大约一半 α'- 和 α- 亚基通过二硫键连接在一起或与 P34 连接。沉降速度实验、尺寸排阻色谱和二维聚丙烯酰胺凝胶电泳 (PAGE) 分析（先进行蓝色非变性 PAGE，然后进行十二烷基硫酸钠-PAGE）表明，含有二硫键连接的 α'/α-亚基的 β-伴大豆球蛋白复合物是超过 720 kD 的复合物。当二硫键与 P34 连接时，α'-和 α-亚基主要以低离子强度存在于大约 480-kD 的复合物（六聚体）中。我们的结果表明，不同β-伴大豆球蛋白六聚体中的α'/α-亚基之间形成二硫键，但P34与α'-和α-亚基的结合减少了β-伴大豆球蛋白六聚体之间的连接。最后，当 β-伴大豆球蛋白在非还原条件下表达时，大豆球蛋白的一个子集被证明以大于六聚体的非共价结合复合物的形式存在。
β-Conglycinin, one of the major soybean (Glycine max) seed storage proteins, is folded and assembled into trimers in the endoplasmic reticulum and accumulated into protein storage vacuoles. Prior experiments have used soybean β-conglycinin extracted using a reducing buffer containing a sulfhydryl reductant such as 2-mercaptoethanol, which reduces both intermolecular and intramolecular disulfide bonds within the proteins. In this study, soybean proteins were extracted from the cotyledons of immature seeds or dry beans under nonreducing conditions to prevent the oxidation of thiol groups and the reduction or exchange of disulfide bonds. We found that approximately half of the α′- and α-subunits of β-conglycinin were disulfide linked, together or with P34, prior to amino-terminal propeptide processing. Sedimentation velocity experiments, size-exclusion chromatography, and two-dimensional polyacrylamide gel electrophoresis (PAGE) analysis, with blue native PAGE followed by sodium dodecyl sulfate-PAGE, indicated that the β-conglycinin complexes containing the disulfide-linked α′/α-subunits were complexes of more than 720 kD. The α′- and α-subunits, when disulfide linked with P34, were mostly present in approximately 480-kD complexes (hexamers) at low ionic strength. Our results suggest that disulfide bonds are formed between α′/α-subunits residing in different β-conglycinin hexamers, but the binding of P34 to α′- and α-subunits reduces the linkage between β-conglycinin hexamers. Finally, a subset of glycinin was shown to exist as noncovalently associated complexes larger than hexamers when β-conglycinin was expressed under nonreducing conditions.