Thelen M P, Delmer D P
ARCO Plant Cell Research Institute, 6560 Trinity Court, Dublin, California 94568.
Plant Physiol. 1986 Jul;81(3):913-8. doi: 10.1104/pp.81.3.913.
We have developed procedures for detection and characterization of UDP-glucose: glucosyltransferases following electrophoretic separation in nondenaturing polyacrylamide gels. Using digitonin-solubilized membrane protein preparations from a variety of plants and two cellulose-producing bacteria, activity can be demonstrated for several UDP-glucose:beta-glucan synthases with an in situ assay following gel electrophoresis. These enzymes can be characterized within the gels with respect to effector requirements and products produced, and several advantages of this assay over solution assays are demonstrated. For example, the clear dependence of plant UDP-glucose:(1-->3)-beta-glucan synthase on both Ca(2+) and a beta-linked glucoside is shown; bacterial cellulose synthases show direct stimulation within the gel by guanyl oligonucleotide, and the Acetobacter xylinum enzyme appears more stable in the gel assay than in solution assay.
我们已开发出在非变性聚丙烯酰胺凝胶中进行电泳分离后检测和鉴定UDP-葡萄糖：葡糖基转移酶的方法。使用来自多种植物和两种产纤维素细菌的洋地黄皂苷增溶膜蛋白制剂，通过凝胶电泳后的原位测定，可以证明几种UDP-葡萄糖：β-葡聚糖合酶的活性。这些酶可以在凝胶中根据效应物需求和产生的产物进行表征，并且证明了该测定法相对于溶液测定法的几个优点。例如，显示出植物UDP-葡萄糖：(1→3)-β-葡聚糖合酶对Ca(2+)和β-连接的糖苷都有明显的依赖性；细菌纤维素合酶在凝胶中受到鸟苷寡核苷酸的直接刺激，并且木醋杆菌酶在凝胶测定中似乎比在溶液测定中更稳定。
