Proteins isolated from lucerne roots by affinity chromatography with sugars analogous to Nod factor moieties.
Proteins isolated from lucerne roots by affinity chromatography with sugars analogous to Nod factor moieties.
复制标题
通过亲和色谱法用类似于 Nod 因子部分的糖从苜蓿根中分离出蛋白质。
DOI：
10.1042/bj3450255
复制
发表时间：
2000
期刊：
影响因子：
--
通讯作者：
S. Brown
中科院分区：
文献类型：
--
作者：
Z. Minić;L. Leproust;J. Laporte;Y. Kouchkovsky;S. Brown
Nod因子是豆科菌共生的重要激发子。这些脂寡糖的任何候选植物受体都可以表现出一些类似凝集素的特性。从紫花苜蓿（lucerne, alfalfa）根的膜组分中，用GlcNAc或N，N‘， N’ -三乙酰-(1- bbb4)- β -d-壳聚糖[(GlcNAc)(3)]接枝到琼脂糖珠作为基质，并在第二步，Sephadex G-200凝胶过滤，分离出一种新的蛋白（P60），一种天然的四聚体，具有60 kDa单体。用（GlcNAc）(3)-琼脂糖分离了一个78 kDa的蛋白。P60对GalNAc、GalN、GlcNAc和GlcN具有特异性血凝活性。与放射性GlcNAc的结合实验得到K(d)为95 nM，每个P60单体有一个结合位点；Nod因子强烈竞争这种结合。在天然PAGE中，与o -硫酸盐Nod因子孵育的蛋白质由于结合而具有更高的电泳迁移率。然而，最大的修饰是Nod因子的自然混合，除了非o -乙酰化的NodRm-IV(S)（单独影响很小）和NodRm-V(S)外，还含有o -乙酰化和o -硫酸化的四糖NodRm-IV(Ac，S)（其中Ac表示非o -乙酰化的基团，S表示还原端的o -硫酸化基团）。原生PAGE研究也对其他来源的已知凝集素进行了研究，但只有美洲商陆（Phytolacca americana）的34 kDa凝集素显示出这种相互作用，尽管Nod因子之间没有区别。最后，对每个分离蛋白的一个肽进行测序；P60的肽段与二氢脂酰胺脱氢酶和铁豆血红蛋白还原酶有一定的相似性，而P78的肽段与70 kDa热休克蛋白的类似区域相同。
Nod factors are important elicitors in legume-bacterium symbiosis. Any candidate plant receptor(s) for these lipo-oligosaccharides can be expected to show some lectin-like properties. A novel protein (P60), a native tetramer with 60 kDa monomers, has been isolated from a membrane fraction of Medicago sativa (lucerne, alfalfa) roots by using affinity chromatography with either GlcNAc or N,N', N"-triacetyl-(1-->4)-beta-d-chitotriose [(GlcNAc)(3)] grafted to agarose beads as the matrix and, in a second step, Sephadex G-200 gel filtration. With (GlcNAc)(3)-agarose an additional protein of 78 kDa was isolated. P60 showed haemagglutination activity with specificity for GalNAc, GalN, GlcNAc and GlcN. Binding experiments with radioactive GlcNAc gave a K(d) of 95 nM and one binding site per monomer of P60; Nod factor competed strongly for this binding. In native PAGE, protein incubated with O-sulphated Nod factors had a higher electrophoretic mobility as a consequence of binding. However, the largest modification was observed with a natural mixture of Nod factors, containing the O-acetylated and O-sulphated tetrasaccharidic NodRm-IV(Ac,S) (in which Ac stands for an O-acetylated group at the non-reducing end and S for O-sulphation at the reducing end) in addition to the non-O-acetylated NodRm-IV(S) (which alone had little effect) and NodRm-V(S). The native PAGE study was also performed with known lectins from other sources, but only the 34 kDa lectin of Phytolacca americana (pokeweed) showed any such interaction, although without discrimination between Nod factors. Finally, one peptide of each isolated protein was sequenced; the peptide from P60 showed some similarity with dihydrolipoamide dehydrogenase and ferric leghaemoglobin reductase, whereas the peptide from P78 was identical with an analogous region of 70 kDa heat shock protein.