Srivastava O P, Srivastava K, Shukla S D
Missouri Lions Eye Research Foundation, Columbia 65201.
Invest Ophthalmol Vis Sci. 1990 Oct;31(10):1982-93.
The pellet recovered after centrifugation (5000 X g) of human corneal endothelial homogenates was used as the source of membranes in these studies. A 66-kilodalton (kD) protein was identified as the most abundant protein in the particulate pellet by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The de novo synthesis of the 66-kD protein by endothelial cells was observed during culturing of human corneas in the presence of 35S-methionine. The 66-kD protein was found to be a plasma membrane protein based on several of its properties, ie, its solubility in CHCl3:CH3OH, its labeling as surface glycoprotein, and during exposure to a photoaffinity hydrophobic probe: 1-azido-4-125I-iodobenzene. Furthermore this protein could be released from the particulate pellet after treatment with phosphatidylinositol-specific phospholipase C, suggesting its anchorage via a phosphatidylinositol glycan linkage in the plasma membrane. Such anchorage of this protein was further confirmed by its labeling during culture of corneas in the presence of 3H-myoinositol. The glycoprotein nature of the 66-kD protein was evident from its labeling during surface glycoprotein labeling of endothelial cells, staining with periodic acid-Schiff stain, and binding to peanut agglutinin (PNA), and lotus agglutinin (LTA) on SDS-acrylamide gels. The 66-kD protein of endothelial particulate pellets recovered from corneas of donors of different ages showed an age-related increase in binding to PNA and LTA. This suggested an increased glycosylation of the 66-kD protein with aging. A polyclonal anti-66-kD protein antibody was used as a probe to determine the presence of this protein in the rabbit and bovine corneal endothelia by the Western-blot analysis. The 66-kD protein was detected in both rabbit and bovine endothelia, but an additional immunoreactive species of 17 kD was also observed which may be a processed product of the 66-kD protein.
在这些研究中，将人角膜内皮匀浆经5000×g离心后回收的沉淀用作膜的来源。通过十二烷基硫酸钠（SDS）-聚丙烯酰胺凝胶电泳，一种66千道尔顿（kD）的蛋白质被鉴定为颗粒沉淀中含量最丰富的蛋白质。在人角膜培养过程中，在存在35S-甲硫氨酸的情况下，观察到内皮细胞从头合成了66-kD蛋白质。基于其若干特性，即其在CHCl3:CH3OH中的溶解性、作为表面糖蛋白的标记以及在暴露于光亲和疏水探针1-叠氮基-4-125I-碘苯期间，发现66-kD蛋白质是一种质膜蛋白。此外，在用磷脂酰肌醇特异性磷脂酶C处理后，该蛋白质可从颗粒沉淀中释放出来，这表明它通过质膜中的磷脂酰肌醇聚糖连接进行锚定。在存在3H-肌醇的情况下，角膜培养期间该蛋白质的标记进一步证实了这种锚定。66-kD蛋白质的糖蛋白性质从内皮细胞表面糖蛋白标记期间的标记、过碘酸-希夫染色以及在SDS-丙烯酰胺凝胶上与花生凝集素（PNA）和莲凝集素（LTA）的结合中显而易见。从不同年龄供体的角膜中回收的内皮颗粒沉淀中的66-kD蛋白质显示，其与PNA和LTA的结合呈年龄相关的增加。这表明随着年龄增长，66-kD蛋白质的糖基化增加。使用多克隆抗66-kD蛋白质抗体作为探针，通过蛋白质印迹分析确定该蛋白质在兔和牛角膜内皮中的存在。在兔和牛内皮中均检测到66-kD蛋白质，但还观察到一种额外的17 kD免疫反应性条带，它可能是66-kD蛋白质的加工产物。
