Gane A M, Craik D, Munro S L, Howlett G J, Clarke A E, Bacic A
Plant Cell Biology Research Centre, School of Botany, University of Melbourne, Parkville, Victoria, Australia.
Carbohydr Res. 1995 Nov 7;277(1):67-85. doi: 10.1016/0008-6215(95)00197-2.
Arabinogalactan-proteins (AGPs) from the female reproductive tissues (stigmas and styles) of Nicotiana alata were isolated from the saturated ammonium sulfate supernatant of buffer-soluble extracts by precipitation with the beta-glucosyl Yariv reagent, followed by gel-filtration chromatography under dissociating conditions. The AGPs had characteristics typical of other AGPs: a high proportion of carbohydrate (95%) with a high ratio of Gal p to Ara f (2:1), and a low protein content (5%) with high levels of alanine, serine, and hydroxyproline. The AGPs consisted of a major species which was almost neutral, and a minor species which was more negatively charged. Sedimentation equilibrium experiments showed that the purified AGPs had a weight-average molecular weight of 143 kD. Linkage analysis showed that the AGPs contained a highly branched backbone of 3-, 6-, and 3,6-linked Gal p residues, bearing terminal Gal p and terminal Ara f residues. Analysis by one-dimensional and two-dimensional 1H and 13C NMR spectroscopy confirmed the presence of these glycosyl linkage types, and showed a high mobility of the terminal Ara f residues consistent with their location on the periphery of the molecules. This analysis represents the most complete 1H assignment for AGP molecules in solution. No difference in the carbohydrate analyses was found between AGPs isolated separately from stigmatic or stylar tissue, or between AGPs isolated from stigmas and styles of plants of different self-incompatibility genotypes.
从烟草的雌性生殖组织（柱头和花柱）中提取阿拉伯半乳聚糖蛋白（AGP），具体方法是先用β-葡萄糖基Yariv试剂沉淀缓冲液可溶性提取物的饱和硫酸铵上清液，然后在解离条件下进行凝胶过滤色谱分离。这些AGP具有其他AGP的典型特征：碳水化合物比例高（95%），Gal p与Ara f的比例高（2:1），蛋白质含量低（5%），丙氨酸、丝氨酸和羟脯氨酸含量高。AGP由一种几乎呈中性的主要成分和一种带更多负电荷的次要成分组成。沉降平衡实验表明，纯化后的AGP重均分子量为143 kD。键合分析表明，AGP含有由3-、6-和3,6-连接的Gal p残基组成的高度分支主链，带有末端Gal p和末端Ara f残基。一维和二维1H和13C NMR光谱分析证实了这些糖基连接类型的存在，并表明末端Ara f残基具有高流动性，这与其在分子外围的位置一致。该分析代表了溶液中AGP分子最完整的1H归属。从柱头或花柱组织中分别分离得到的AGP之间，或从不同自交不亲和基因型植物的柱头和花柱中分离得到的AGP之间，在碳水化合物分析上没有发现差异。
