Dorne A J, Joyard J, Block M A, Douce R
J Cell Biol. 1985 May;100(5):1690-7. doi: 10.1083/jcb.100.5.1690.
We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes.
我们研究了蜡样芽孢杆菌的磷脂酶C对包膜膜囊泡和完整叶绿体中磷脂水解程度的影响。当分离的包膜囊泡在磷脂酶C存在的情况下孵育时，如果磷脂酰胆碱和磷脂酰甘油能被该酶作用（即当囊泡在磷脂酶C存在的情况下进行超声处理），它们会完全转化为二酰基甘油，但磷脂酰肌醇不会。这些实验表明，磷脂酶C可用于探测菠菜叶绿体外部包膜膜胞质侧磷脂酰胆碱和磷脂酰甘油的可及性。当分离、纯化的完整叶绿体在非常温和的条件下（12℃孵育1分钟）与少量磷脂酶C（0.3 U/mg叶绿素）孵育时，超过80%的磷脂酰胆碱分子被水解，而几乎没有磷脂酰甘油分子被水解。由于我们还通过几种不同的方法（相差显微镜和电子显微镜、免疫化学和电泳分析）证明，菠菜叶绿体，尤其是其外部包膜膜，在用磷脂酶C进行温和处理后仍保持完整，我们可以得出结论，菠菜叶绿体外部包膜膜上的磷脂存在明显的不对称分布。磷脂酰胆碱是外部包膜膜的主要极性脂质，几乎完全可从膜胞质侧接触到，因此可能定位在外部包膜双层的外层小叶中。相反，磷脂酰甘油是内部包膜膜和类囊体中的主要极性脂质，可能无法从胞质中被磷脂酶C作用，因此可能主要定位在外部包膜膜的内层小叶和其他叶绿体膜中。
