Joyard J, Billecocq A, Bartlett S G, Block M A, Chua N H, Douce R
J Biol Chem. 1983 Aug 25;258(16):10000-6.
Nonpenetrating proteolytic enzymes (such as thermolysin) were used to probe the cytosolic surface of the outer envelope membrane from spinach chloroplasts. Up to 20 different envelope polypeptides were susceptible to a mild digestion of isolated intact chloroplasts by thermolysin. Most of the thermolysin-sensitive envelope polypeptides were not extracted by a mixture of chloroform/methanol (2:1, v/v). A clear exception was E10 which is hydrophobic and, in addition, is an integral membrane polypeptide. Using antibodies to envelope polypeptides sensitive (E10 and E24) and insensitive (E30 and E37) to thermolysin, we demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, induced agglutination of intact chloroplasts. In addition, immunofluorescence experiments demonstrated that only antibodies to E10 and E24, but not antibodies to E30 and E37, gave a green fluorescence at the outer surface of intact chloroplasts. These experiments demonstrate that E10 and E24, and probably all the thermolysin-sensitive envelope polypeptides, are accessible from the cytosolic side of the outer membrane of the chloroplast envelope.
非穿透性蛋白水解酶（如嗜热菌蛋白酶）被用于探测菠菜叶绿体外被膜的胞质表面。多达20种不同的外被膜多肽会因嗜热菌蛋白酶对分离的完整叶绿体进行温和消化而变得敏感。大多数对嗜热菌蛋白酶敏感的外被膜多肽不能被氯仿/甲醇（2:1，v/v）混合物提取出来。一个明显的例外是E10，它具有疏水性，此外还是一种整合膜多肽。使用针对对嗜热菌蛋白酶敏感（E10和E24）和不敏感（E30和E37）的外被膜多肽的抗体，我们证明只有针对E10和E24的抗体，而不是针对E30和E37的抗体，能诱导完整叶绿体的凝集。此外，免疫荧光实验表明只有针对E10和E24的抗体，而不是针对E30和E37的抗体，能在完整叶绿体的外表面产生绿色荧光。这些实验表明E10和E24，可能还有所有对嗜热菌蛋白酶敏感的外被膜多肽，都可以从叶绿体被膜外膜的胞质侧接触到。
