High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors.
High affinity binding of a glycopeptide elicitor to tomato cells and microsomal membranes and displacement by specific glycan suppressors.
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糖肽激发子与番茄细胞和微粒体膜的高亲和力结合，并被特定聚糖抑制子取代。
DOI：
10.1016/s0021-9258(18)82393-6
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发表时间：
1993
期刊：
影响因子：
--
通讯作者：
Thomas BollerQ
中科院分区：
文献类型：
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作者：
Christoph W. BasseS;Angelika Fath;Thomas BollerQ
我们先前已经分离了来源于酵母转化酶的糖肽，其在悬浮培养的番茄细胞中充当高效诱导子，诱导乙烯生物合成和苯丙氨酸解氨酶活性，并且我们已经发现通过内切-β-N-乙酰氨基葡糖苷酶H从纯糖肽诱导子释放的高甘露糖寡糖充当诱导子活性的抑制子（Basse，C. W.，Bock，K.，和Boller，T.（1992）J.Biol.Chem.267，10258-10265）。其中一种激发子活性糖肽（gp 8 c）用叔丁氧羰基-L-[35 S]甲硫氨酸标记，并通过反相高效液相色谱法纯化，导致衍生物的比放射性约为900 Ci/mmol。这种放射性标记的糖肽表现出特定的，饱和的，和可逆的结合，整个番茄细胞的条件下，细胞响应激发子以及来自这些细胞的微粒体膜。配体饱和实验，与微粒体膜进行，给出了一个解离常数（Kd）为3.3 nM的Scatchard分析确定。各种糖肽激发子和酵母转化酶制剂进行了比较，他们的能力，竞争结合35 S-标记的糖蛋白8 C番茄膜和诱导乙烯在番茄细胞的生物合成。这些研究揭示了体内激发子活性与体外置换活性之间的高度相关性。在这两个测试中，高活性取决于由多于8个甘露糖残基组成的聚糖侧链的存在。高甘露糖低聚糖在体内作为激发子活性的抑制剂，也竞争标记的激发子的结合。抑制剂活性聚糖Man 11 GlcNAc和激发剂活性gp 8 c表现出非常相似的置换活性，并且聚糖Man 11 GlcNAc的抑制常数（Ki）与35 S标记gp 8 c计算的Kd值非常相似，表明糖肽激发剂和来自这些激发剂的聚糖抑制剂以相似的亲和力竞争相同的结合位点。与抑制剂活性聚糖Man 11 GlcNAc相比，抑制剂非活性聚糖Man 8 GlcNAc竞争35 S标记的gp 8 c与番茄膜结合的能力低200倍。我们的研究结果表明，番茄细胞膜中存在一个特定的激发子结合位点，并表明糖肽和聚糖作为激动剂和拮抗剂诱导的应激反应，分别通过竞争这个结合位点。
We have previously isolated glycopeptides derived from yeast invertase that acted as highly potent elicitors in suspension-cultured tomato cells, inducing ethylene biosynthesis and phenylalanine ammonia-lyase activity, and we have found that the high mannose oligosaccharides released from the pure glycopeptide elicitors by endo-beta-N-acetylglucosaminidase H acted as suppressors of elicitor activity (Basse, C. W., Bock, K., and Boller, T. (1992) J. Biol. Chem. 267, 10258-10265). One of the elicitor-active glycopeptides (gp 8c) was labeled with t-butoxycarbonyl-L-[35S]methionine and purified by reversed phase high performance liquid chromatography resulting in a specific radioactivity of the derivative of about 900 Ci/mmol. This radiolabeled glycopeptide showed specific, saturable, and reversible binding to whole tomato cells under conditions in which cells are responsive to elicitors as well as to microsomal membranes derived from these cells. Ligand saturation experiments, performed with microsomal membranes, gave a dissociation constant (Kd) of 3.3 nM as determined by Scatchard analysis. Various glycopeptide elicitors and preparations from yeast invertase were compared with respect to their abilities to compete for binding of 35S-labeled gp 8c to tomato membranes and to induce ethylene biosynthesis in tomato cells. These studies revealed a high degree of correlation between elicitor activities in vivo and displacement activities in vitro. In both tests, a high activity depended on the presence of glycan side chains consisting of more than 8 mannosyl residues. The high mannose oligosaccharides that acted as suppressors of elicitor activity in vivo competed for binding of the labeled elicitor also. The suppressor-active glycan Man11GlcNAc and the elicitor-active gp 8c exhibited very similar displacement activities, and the inhibitory constant (Ki) of the glycan Man11GlcNAc was very similar to the Kd value calculated for 35S-labeled gp 8c, indicating that the glycopeptide elicitors and the glycan suppressors derived from these elicitors competed with similar affinities for the same binding site. The suppressor-inactive glycan Man8GlcNAc had a 200-fold lower capacity to compete for binding of 35S-labeled gp 8c to tomato membranes compared with the suppressor-active glycan Man11GlcNAc. Our results demonstrate the existence of a specific elicitor binding site in tomato cell membranes and suggest that glycopeptides and glycans act as agonists and antagonists for induction of the stress response, respectively, by competing for this binding site.