Plant Growth Laboratory, University of California, Davis, California 95616.
Plant Physiol. 1984 May;75(1):70-3. doi: 10.1104/pp.75.1.70.
The localization of two previously characterized endoproteinases (EP(1) and EP(2)) that comprise more than 95% of the protease activity in primary Hordeum vulgare L. var Numar leaves was determined. Intact vacuoles released from washed mesophyll protoplasts by gentle osmotic shock and increase in pH, were purified by flotation through a four-step Ficoll gradient. These vacuoles contained endoproteinases that rapidly degraded purified barley ribulose-1,5-bisphosphate carboxylase (RuBPCase) substrate. Breakdown products and extent of digestion of RuBPCase were determined using 12% polyacrylamide-sodium dodecyl sulfate gels. Coomassie brilliant blue- or silver-stained gels were scanned, and the peaks were integrated to provide quantitative information. The characteristics of the vacuolar endoproteinases (e.g. sensitivity to various inhibitors and activators, and the molecular weights of the breakdown products, i.e. peptide maps) closely resembled those of purified EP(1) and partially purified EP(2). It is therefore concluded that EP(1) and EP(2) are localized in the vacuoles of mesophyll cells.
两种先前被鉴定的内肽酶（EP(1)和 EP(2)）在大麦叶片原生质体中占蛋白酶活性的 95%以上，我们对其进行了定位研究。通过温和的渗透压冲击和 pH 值升高，从清洗过的叶肉原生质体中释放出完整的液泡，然后通过四步菲可尔梯度浮选进行纯化。这些液泡中含有内肽酶，可迅速降解纯化的大麦核酮糖-1,5-二磷酸羧化酶（RuBPCase）底物。使用 12%聚丙烯酰胺-十二烷基硫酸钠凝胶来确定 RuBPCase 的降解产物和消化程度。考马斯亮蓝或银染凝胶扫描后，对峰进行积分，以提供定量信息。液泡内肽酶的特性（例如对各种抑制剂和激活剂的敏感性以及降解产物的分子量，即肽图）与纯化的 EP(1)和部分纯化的 EP(2)非常相似。因此，可以得出结论，EP(1)和 EP(2)定位于叶肉细胞的液泡中。
