Department of Botany and Plant Sciences, University of California, Riverside, California 92521.
Plant Physiol. 1986 Jul;81(3):869-74. doi: 10.1104/pp.81.3.869.
Proteolytic activity was detected, using a sensitive radial diffusion plate assay, in the plasma membrane fractions of corn (Zea mays L.) roots and from roots of several other plant species. The proteases could be effectively inhibited in corn with phenylmethane sulfonyl fluoride or chymostatin. Protease activity of oat roots, however, was not significantly reduced by these inhibitors. The results of diffusion plate assay were confirmed with the less sensitive azocasein assay using crude cell homogenates. Chymostatin and phenylmethane sulfonyl fluoride were effective in preventing protease degradation of polypeptides as revealed by electrophoresis. The diffusion plate assay uses a permanent support for a 0.75 millimeter thick agarose slab containing 200 micrograms per milliliter casein. By staining the fixed and dried gel with Coomassie blue R-250, proteolytic activity was visualized as a cleared area around the sample well with a detection limit of about 0.3 nanograms trypsin. The diffusion plate assay should prove useful for screening inhibitors of proteases where limited amounts of material are available, such as with plant cell fractions or highly purified proteins.
用灵敏的放射状扩散平板法检测到玉米（Zea mays L.）根的质膜部分和其他几种植物根中有蛋白水解酶活性。用苯甲基磺酰氟或抑酶肽可以有效地抑制玉米中的蛋白酶。然而，这些抑制剂对燕麦根中的蛋白酶活性没有明显的抑制作用。扩散平板法的结果用粗细胞匀浆的敏感性较低的偶氮酪蛋白法得到证实。用电泳法揭示，抑酶肽和苯甲基磺酰氟能有效地防止蛋白酶对多肽的降解。扩散平板法使用永久性支架，支架上有 0.75 毫米厚的琼脂糖平板，每毫升含有 200 微克酪蛋白。用考马斯亮蓝 R-250 固定和干燥凝胶后，蛋白酶活性在样品孔周围呈现为清晰区域，检测限约为 0.3 纳克胰蛋白酶。扩散平板法应该对筛选蛋白酶抑制剂很有用，因为这种方法只需很少的材料，如植物细胞部分或高度纯化的蛋白质。
