McCarthy K J, Accavitti M A, Couchman J R
Department of Cell Biology and Anatomy, University of Alabama, Birmingham 35294.
J Cell Biol. 1989 Dec;109(6 Pt 1):3187-98. doi: 10.1083/jcb.109.6.3187.
Reichert's membrane, an extraembryonic membrane present in developing rodents, has been proposed as an in vivo model for the study of basement membranes. We have used this membrane as a source for isolation of basement membrane proteoglycans. Reichert's membranes were extracted in a guanidine/3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate buffer followed by cesium chloride density-gradient ultracentrifugation under dissociative conditions. The proteoglycans were subsequently purified from the two most dense fractions (greater than 1.3 g/ml) by ion-exchange chromatography. Mice were immunized with the proteoglycan preparation and four mAbs recognizing the core protein of a high-density, buoyant chondroitin sulfate proteoglycan were raised. Confirmation of antibody specificity was carried out by the preparation of affinity columns made from each of the mAbs. Chondroitin sulfate proteoglycans (CSPGs) were purified from both supernatant and tissue fractions of Reichert's membranes incubated in short-term organ culture in the presence of radiolabel. The resultant affinity-purified proteoglycan samples were examined by gel filtration, SDS-PAGE, and immunoblotting. This proteoglycan is of high molecular weight (Mr = 5-6 x 10(5)), with a core protein of Mr = approximately 1.5-1.6 x 10(5) and composed exclusively of chondroitin sulfate chains with an average Mr = 1.6-1.8 x 10(4). In addition, a CSPG was purified from adult rat kidney, whose core protein was also Mr = 1.6 x 10(5). The proteoglycan and its core protein were also recognized by all four mAbs. Indirect immunofluorescence of rat tissue sections stained with these antibodies reveal a widespread distribution of this proteoglycan, localized specifically to Reichert's membrane and nearly all basement membranes of rat tissues. In addition to heparan sulfate proteoglycans, it therefore appears that at least one CSPG is a widespread basement membrane component.
雷歇特氏膜是发育中的啮齿动物体内存在的一种胚外膜，已被提议作为研究基底膜的体内模型。我们已将此膜用作分离基底膜蛋白聚糖的来源。雷歇特氏膜在胍/3-[(3-胆酰胺丙基)二甲基铵]-1-丙烷磺酸盐缓冲液中提取，随后在解离条件下进行氯化铯密度梯度超速离心。随后通过离子交换色谱从两个密度最高的级分（大于1.3 g/ml）中纯化蛋白聚糖。用该蛋白聚糖制剂免疫小鼠，制备了四种识别高密度、漂浮硫酸软骨素蛋白聚糖核心蛋白的单克隆抗体。通过制备由每种单克隆抗体制成的亲和柱来确认抗体特异性。在放射性标记存在的情况下，从短期器官培养中孵育的雷歇特氏膜的上清液和组织级分中纯化硫酸软骨素蛋白聚糖（CSPG）。通过凝胶过滤、SDS-PAGE和免疫印迹检查所得的亲和纯化蛋白聚糖样品。这种蛋白聚糖分子量高（Mr = 5 - 6×10⁵），核心蛋白Mr约为1.5 - 1.6×10⁵，仅由平均Mr = 1.6 - 1.8×10⁴的硫酸软骨素链组成。此外，从成年大鼠肾脏中纯化出一种CSPG，其核心蛋白Mr也为1.6×10⁵。所有四种单克隆抗体也识别该蛋白聚糖及其核心蛋白。用这些抗体对大鼠组织切片进行间接免疫荧光显示，这种蛋白聚糖分布广泛，特异性定位于雷歇特氏膜和大鼠组织的几乎所有基底膜。因此，除了硫酸乙酰肝素蛋白聚糖外，似乎至少有一种CSPG是广泛存在的基底膜成分。
