Agriculture Canada Research Station, Winnipeg, Manitoba, Canada R3T 2M9.
Plant Physiol. 1985 Nov;79(3):820-4. doi: 10.1104/pp.79.3.820.
Proteins in intercellular washing fluid (IWF) from noninoculated and stem rust-affected wheat leaves were separated by isoelectric focusing and polyacrylamide gel electrophoresis under nondenaturing conditions, transferred to nitrocellulose membranes, and assayed in situ for peroxidase and glycosidase activity.Two infection-related peroxidase isozymes were detected in addition to more than ten that were present only in noninoculated plants. One other peroxidase isozyme was present in much higher concentration in IWF from infected leaves than in IWF from noninoculated leaves.IWF contained many polymorphic glycosidases. A new method is described to localize the glycosidase isozymes accurately on nitrocellulose blots for evaluation of their substrate specificities: each blot was developed with the appropriate p-nitrophenyl glycoside to reveal glycosidase activity, then reprobed for concanavalin A-binding glycoproteins to serve as an internal reference frame for blot-to-blot comparisons. This procedure also provided information on glycosylation of the isozymes.The locations of at least 15 (out of 37) isozymes were coincident with concanavalin A binding, including those of all 10 alpha-d-mannosidases, and of 6 beta-d-xylosidases. On five areas of the blots there was coincidence of beta-d-xylosidase and alpha-l-arabinosidase activity. Three of these areas corresponded to three of the most prominent Coomassie brilliant blue-stainable IWF proteins in gels. Isozymes that could convert p-nitrophenyl-beta-d-glucoside, -beta-d-galactoside, and/or -beta-d-fucoside revealed a complex pattern of partially overlapping substrate specificities: two isozymes utilized both glucose and fucose derivatives, one utilized all three derivatives, and several others converted only one of the three substrates. No enzymes were detected with activity on p-nitrophenyl-beta-d-galactosaminide, -beta-l-fucoside, or -alpha-d-galactoside. No additional glycosidases were detected in IWF from stem rust-affected leaves.
非接种和感染叶锈病的小麦叶片细胞间洗液（IWF）中的蛋白质在非变性条件下通过等电聚焦和聚丙烯酰胺凝胶电泳分离，转移到硝酸纤维素膜上，并在原位检测过氧化物酶和糖苷酶活性。除了存在于非接种植物中的十多种外，还检测到两种与感染相关的过氧化物酶同工酶。感染叶片的 IWF 中过氧化物酶同工酶的浓度明显高于非接种叶片的 IWF。IWF 中含有许多多态性糖苷酶。本文描述了一种新的方法，可以准确地将糖苷酶同工酶定位在硝酸纤维素印迹上，以评估它们的底物特异性：每个印迹用适当的 p-硝基苯糖苷进行开发，以显示糖苷酶活性，然后用伴刀豆球蛋白 A 结合糖蛋白重新探测，作为印迹间比较的内部参考框架。该程序还提供了同工酶糖基化的信息。至少有 15 种（37 种中的 15 种）同工酶的位置与伴刀豆球蛋白 A 结合，包括 10 种α-d-甘露糖苷酶和 6 种β-d-木糖苷酶。在印迹的五个区域，β-d-木糖苷酶和α-l-阿拉伯糖苷酶活性存在重合。这些区域中的三个与凝胶中三种最显著的考马斯亮蓝染色 IWF 蛋白相对应。能够转化 p-硝基苯-β-d-葡萄糖苷、-β-d-半乳糖苷和/或-β-d-岩藻糖苷的同工酶显示出部分重叠的底物特异性的复杂模式：两种同工酶利用葡萄糖和岩藻糖衍生物，一种同工酶利用所有三种衍生物，还有几种同工酶只转化三种底物中的一种。没有检测到对 p-硝基苯-β-d-半乳糖胺、-β-l-岩藻糖苷或-α-d-半乳糖苷有活性的酶。在感染叶锈病的叶片的 IWF 中未检测到其他糖苷酶。
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