Fermin C D, Martin D S, Li Y T, Li S C
Department of Pathology, Tulane University School of Medicine, New Orleans, LA 70112-2699, USA.
Cell Mol Biol (Noisy-le-grand). 1995 Jun;41(4):577-91.
The otoconial matrix (OM) of chicks (Gallus domesticus) inner ear was analyzed. Histochemically the OM was reacted with phosphotungstic acid (PTA) and immunohistochemically with the monoclonal antibody antikeratan sulfate (antiKS). The OM was digested with the enzyme endo-beta-galactosidase (E beta Galase) or separated by 1D and 2D gel electrophoresis. PTA which reacts with glycoproteins precipitated the OM, suggesting that the OM contains glycoproteins. A central core in each crystal had no PTA staining, suggesting that the core lacked glycoproteins. Anti KS antibody stained the OM with increased density in older embryos as determined by color thresholding. E beta Galase, which cleaves the lactosamine repeating units in KS, decreased the immunostain by 30% in the OM and by 20% in the cartilage. The OM from the utricle, saccule and macula lagena contained similar molecular weight bands. Five dense bands in the OM were less dense in tissue and blood controls, suggesting that such bands are enriched in the OM. Isoelectric focusing of the OM showed a negatively charged high molecular weight smear not present in blood and faint in tissue controls. The high affinity of the OM for the cationic PTA stain, the strong immunohistochemical reaction of the OM with anti KS antibody and high molecular weight negative smear in 2D gels taken together suggest that: a) the OM contains large amounts of glycoproteins and glycans, one of which is keratan sulfate, because its immuno stain with antiKS antibody was decreased by the enzyme E beta Galase, b) the utricle, saccule and macula lagena may have similar composition, and c) the concentration of KS may increase gradually until complete mineralization of the OM is reached.
对家鸡（Gallus domesticus）内耳的耳石基质（OM）进行了分析。组织化学上，OM与磷钨酸（PTA）反应，免疫组织化学上与抗硫酸角质素单克隆抗体（antiKS）反应。用内切β-半乳糖苷酶（EβGalase）消化OM或通过一维和二维凝胶电泳分离OM。与糖蛋白反应的PTA使OM沉淀，表明OM含有糖蛋白。每个晶体的中心核没有PTA染色，表明该核缺乏糖蛋白。通过颜色阈值测定，anti KS抗体在较老胚胎中对OM的染色密度增加。EβGalase可切割KS中的乳糖胺重复单元，使OM中的免疫染色降低30%，软骨中的免疫染色降低20%。来自椭圆囊、球囊和壶腹嵴的OM含有相似分子量的条带。OM中的五条致密条带在组织和血液对照中较不致密，表明这些条带在OM中富集。OM的等电聚焦显示在血液中不存在且在组织对照中微弱的带负电荷的高分子量拖尾。OM对阳离子PTA染色的高亲和力、OM与anti KS抗体的强烈免疫组织化学反应以及二维凝胶中的高分子量负拖尾共同表明：a）OM含有大量糖蛋白和聚糖，其中之一是硫酸角质素，因为其与antiKS抗体的免疫染色被EβGalase酶降低；b）椭圆囊、球囊和壶腹嵴可能具有相似的组成；c）KS的浓度可能会逐渐增加，直到OM完全矿化。
