Educational Laboratory of Fundamental Biology for Pharmacology, Showa Pharmaceutical University, Machida, Tokyo, 194-8543, Japan.
Physiol Plant. 2018 Jun;163(2):259-266. doi: 10.1111/ppl.12687. Epub 2018 Mar 13.
To understand the action mechanism of yieldin (YLD) on the regulation of the yield threshold (Y), one of the critical parameters of cell wall extension, YLD was extracted from the cell walls of cowpea (Vigna unguiculata L.) hypocotyls and the hemagglutinin activity (HA) as well as the glycosidase activity of the protein was measured. Sedimentation assays using trypsinated rabbit erythrocytes showed that YLD possessed HA at pH 7. The digestion assays using 4-nitrophenyl (pNP) glycopyranosides as artificial substrates showed that YLD liberated galactose residues from pNP alpha-d-galactopyranoside mainly at pH 4.0, i.e. the pH level where Y was decreased at most. These results show that YLD is a bifunctional protein that switches between the HA and the galactosidase activities depending on the surrounding pH. Since D-galactose at concentration of 0.03 g l perfectly inhibited the HA, YLD was suggested to associate with galactose residues. However, the galactose application ten times concentrated was necessary to inhibit both the galactosidase activity of YLD and the acid-induced shift of Y regulated by YLD. In addition, the specific inhibitor of alpha-d-galactosidase (deoxygalactonojirimycin) inhibited both the galactosidase activity of YLD and the shift of Y at the same concentration, but not the HA. On the basis of these results, it is suggested the galactosidase activity of YLD plays a central role in the mechanism of Y-regulation at acidic pH.
为了理解膨压素（YLD）对细胞壁延伸关键参数之一的伸长阈（Y）调节的作用机制，从豇豆花叶下胚轴细胞壁中提取 YLD，并测定其血凝素活性（HA）和糖苷酶活性。使用胰蛋白酶处理的兔血红细胞的沉降测定表明，YLD 在 pH 7 时具有 HA。使用 4-硝基苯（pNP）糖苷作为人工底物的消化测定表明，YLD 主要在 pH 4.0 时从 pNP α-D-半乳糖吡喃糖苷中释放半乳糖残基，即 Y 降低最多的 pH 水平。这些结果表明，YLD 是一种具有两种功能的蛋白质，根据周围 pH 值在 HA 和半乳糖苷酶活性之间切换。由于 0.03 g l 的 D-半乳糖完全抑制了 HA，因此推测 YLD 与半乳糖残基结合。然而，需要浓缩 10 倍的半乳糖才能抑制 YLD 的半乳糖苷酶活性和 YLD 调节的酸性诱导的 Y 移位。此外，α-D-半乳糖苷酶的特异性抑制剂（脱氧半乳糖酸脒）以相同的浓度抑制 YLD 的半乳糖苷酶活性和 Y 的移位，但不抑制 HA。基于这些结果，表明 YLD 的半乳糖苷酶活性在酸性 pH 下 Y 调节的机制中起核心作用。
