Vargas F F, Osorio H M, Basilio C, De Jesus M, Ryan U S
Division of Cardio-Renal Drug Products, ODEI, CDER, Food and Drug Administration, Bethesda, MD.
Membr Biochem. 1990 Apr-Jun;9(2):83-9. doi: 10.3109/09687689009025831.
The main purpose of this work was to identify the macromolecules carrying the surface charge of endothelial cells. This was done by measuring changes in cell electrophoretic mobility caused by enzymatic removal of glycocalyx components. Endothelial cells were removed from the bovine pulmonary artery using nonenzymatic procedures, plated, and identified by immunocytochemical methods and electron microscopy. Cultured cells were suspended in saline and placed in the lumen of a capillary in a Rank Brothers electrophoresis instrument. Voltage was applied between the ends of the capillary, and the velocity acquired by the cells was measured with a microscope. Preincubating the cells in protein-free saline for 1 h reduced the mobility by 25%. This reflects the loss of proteoheparan sulfate from the cell surface. Cell mobility was totally suppressed by exposing the entire cell surface to chondroitin sulfate lyase, but it was only slightly diminished when the enzyme was applied only to the cell side facing the culture medium. A partial decrease in mobility was obtained after enzymatic removal of either heparin, heparan sulfate, or collagen. The results indicate that sulfated glycosaminoglycans are the main carriers of the surface change in vascular endothelial cells. The asymmetrical effect of chondroitinase on the two sides of the cell indicates a distribution polarization for glycosaminoglycans in endothelial cells.
这项工作的主要目的是鉴定携带内皮细胞表面电荷的大分子。通过测量因酶促去除糖萼成分而导致的细胞电泳迁移率变化来实现这一目的。使用非酶法从牛肺动脉中分离出内皮细胞，进行铺板，并通过免疫细胞化学方法和电子显微镜进行鉴定。将培养的细胞悬浮在盐溶液中，置于Rank Brothers电泳仪的毛细管管腔中。在毛细管两端施加电压，并用显微镜测量细胞获得的速度。将细胞在无蛋白盐溶液中预孵育1小时后，迁移率降低了25%。这反映了细胞表面蛋白聚糖硫酸乙酰肝素的丢失。当整个细胞表面暴露于硫酸软骨素酶时，细胞迁移率完全受到抑制，但当该酶仅作用于面向培养基的细胞一侧时，迁移率仅略有降低。酶促去除肝素、硫酸乙酰肝素或胶原蛋白后，迁移率出现部分下降。结果表明，硫酸化糖胺聚糖是血管内皮细胞表面电荷的主要载体。硫酸软骨素酶对细胞两侧的不对称作用表明糖胺聚糖在内皮细胞中的分布存在极化现象。
