Campargue C, Lafitte C, Esquerré-Tugayé M T, Mazau D
Centre de Biologie et Physiologie Végétales, UMR CNRS, UPS 5546, Université Paul Sabatier, Toulouse, France.
Anal Biochem. 1998 Mar 1;257(1):20-5. doi: 10.1006/abio.1997.2526.
The plant cell wall hydroxyproline-rich glycoprotein (HRGP), also called extensin, contains arabinose and oligoarabinoside side chains O-glycosidically linked to hydroxyproline (Hyp). We present a highly sensitive method for determining both the glycosylation pattern and the Hyp content of HRGP requiring only nanomole amounts of each Hyp-compound for accurate determination. This method is based on anion-exchange chromatography followed by pulsed amperometric detection of the Hyp-oligoarabinosides and Hyp released from HRGP by 0.22 M Ba(OH)2 hydrolysis, which cleaves only peptidyl bonds. A sodium acetate gradient (0-250 mM) in 150 mM NaOH elutes Hyp and the Hyp-oligoarabinosides Hyp-(Ara)1-5 in less than 40 min. We have used this procedure to determine the glycosylation pattern of Hyp in plant cell walls, without prior isolation of HRGP.
植物细胞壁富含羟脯氨酸的糖蛋白（HRGP），也称为伸展蛋白，含有通过O-糖苷键与羟脯氨酸（Hyp）相连的阿拉伯糖和低聚阿拉伯糖苷侧链。我们提出了一种高度灵敏的方法，用于测定HRGP的糖基化模式和Hyp含量，准确测定每种Hyp化合物仅需纳摩尔量。该方法基于阴离子交换色谱，随后通过脉冲安培检测经0.22 M Ba(OH)₂水解从HRGP释放的Hyp-低聚阿拉伯糖苷和Hyp，0.22 M Ba(OH)₂仅切割肽键。在150 mM NaOH中用乙酸钠梯度（0 - 250 mM）洗脱Hyp和Hyp-低聚阿拉伯糖苷Hyp-(Ara)1-5，洗脱时间不到40分钟。我们已使用该程序来确定植物细胞壁中Hyp的糖基化模式，无需事先分离HRGP。
