Laurie G W, Inoue S, Bing J T, Hassell J R
Laboratory of Developmental Biology and Anomalies, National Institute of Dental Research, Bethesda, Maryland 20892.
Am J Anat. 1988 Mar;181(3):320-6. doi: 10.1002/aja.1001810308.
Kleinschmidt spreading, negative staining, and rotary shadowing were used to examine the large form of (basement membrane) heparan sulfate proteoglycan in the electron microscope. Heparan sulfate proteoglycan was visualized as consisting of two parts: the core protein and, emerging from one end of the core protein, the glycosaminoglycan side chains. The core protein usually appeared as an S-shaped rod with about six globules along its length. Similar characteristics were observed in preparations of core protein in which the side chains had been removed by heparitinase treatment ("400-kDa core") as well as in a 200-kDa trypsin fragment ("P200") derived from one end of the core protein. The core protein was sensitive to lyophilization and apparently also to the method of examination, being condensed following Kleinschmidt spreading (length means = 52 nm) and extended following negative staining (length means = 83 nm) or rotary shadowing (length means = 87 nm; 400-kDa core length means = 80 nm; P200 length means = 44 nm). Two or three glycosaminoglycan side chains (length means = 146 +/- 53 nm) were attached to one end of the core protein. The side chains often appeared tangled or to merge together as one. Thus, the large heparan sulfate proteoglycan from basement membrane is an asymmetrical molecule with a core protein containing globular domains and terminally attached side chains. This structure is in keeping with that previously predicted by enzymatic digestions and with the proposed orientation in basement membranes, i.e., the core protein bound in the lamina densa and the heparan sulfate side chains in the lamina lucida arranged along the surface of the basement membranes.
采用克莱因施密特铺展法、负染色法和旋转投影法在电子显微镜下检测（基底膜）硫酸乙酰肝素蛋白聚糖的大分子形式。硫酸乙酰肝素蛋白聚糖表现为由两部分组成：核心蛋白以及从核心蛋白一端伸出的糖胺聚糖侧链。核心蛋白通常呈现为S形杆状，沿其长度方向约有六个球状结构。在经肝素酶处理去除侧链的核心蛋白制剂（“400 kDa核心蛋白”）以及源自核心蛋白一端的200 kDa胰蛋白酶片段（“P200”）中也观察到了类似特征。核心蛋白对冻干敏感，显然也受检测方法影响，在克莱因施密特铺展后会浓缩（长度平均值 = 52 nm），而在负染色（长度平均值 = 83 nm）或旋转投影（长度平均值 = 87 nm；400 kDa核心蛋白长度平均值 = 80 nm；P200长度平均值 = 44 nm）后会伸展。两到三条糖胺聚糖侧链（长度平均值 = 146 ± 53 nm）连接在核心蛋白的一端。这些侧链常常看起来相互缠绕或融合成一条。因此，来自基底膜的大分子硫酸乙酰肝素蛋白聚糖是一种不对称分子，其核心蛋白含有球状结构域并带有末端连接的侧链。这种结构与先前通过酶消化预测的结构以及在基底膜中提出的取向一致，即核心蛋白结合在致密层中，而硫酸乙酰肝素侧链在透明层中沿基底膜表面排列。
