Platelet glycocalicin. Its membrane association and solubilization in aqueous media.
Platelet glycocalicin. Its membrane association and solubilization in aqueous media.
复制标题
血小板糖运载蛋白。
DOI：
10.1016/0005-2736(80)90102-9
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发表时间：
1980
期刊：
影响因子：
--
通讯作者：
Torun Stabaek
中科院分区：
文献类型：
--
作者：
N. Solum;I. Hagen;C. Filion;Torun Stabaek
用3MKCl从人血小板中提取糖钙素，并用Sepharose偶联麦芽凝集素亲和层析柱进行纯化是最有效的步骤。纯化蛋白的兔抗血清能凝集人血小板，并能抑制牛凝血因子VIII相关蛋白诱导的凝集。在含有Triton X-100的琼脂糖凝胶中，血小板Triton X-100提取物的交叉免疫电泳法显示存在两种不同电泳率的糖钙蛋白相关组分，与该抗血清形成连续的双峰免疫沉淀。快速移动组分代表免疫沉淀物的次峰，对应于纯化的可溶性糖钙素。交叉疏水相互作用免疫电泳法未显示纯化的糖钙素或快速移动组分作为疏水基质与苯基-琼脂糖凝胶CL-4B结合。慢速组分代表免疫沉淀物的主峰，显示出与疏水基质的强烈结合。免疫电泳法测定水介质中糖钙蛋白的存在表明，EDTA、N-乙基马来酰亚胺和碘乙酰胺的存在显著降低了糖钙蛋白的增溶作用。在相同浓度下，这些抑制剂强烈抑制血小板超声波钙激活的蛋白水解酶。酸解后的唾液酸测定表明，在没有EDTA的情况下裂解时，其唾液酸的含量明显高于在EDTA存在下的裂解，糖钙素对总唾液酸的贡献很大。
Glycocalicin has been extracted from human platelets by 3 M KCl and purified using affinity chromatography on columns of Sepharose-coupled wheat germ agglutinin as the most efficient step. Rabbit antiserum to the purified protein agglutinated human platelets and inhibited the agglutination induced by bovine Factor VIII-related protein. Crossed immunoelectrophoresis of Triton X-100 extracts of platelets in Triton X-100-containing agarose revealed the presence of two glycocalicin-related components of different electrophoretic mobilities giving a continuous double-peak immunoprecipitate with this antiserum. The fast-moving component, which represented the minor peak of the immunoprecipitate, corresponded to purified soluble glycocalicin. Crossed hydrophobic interaction immunoelectrophoresis did not demonstrate binding of the purified glycocalicin or the fast-moving component to phenyl-Sepharose CL-4B as hydrophobic matrix. The slow-moving component, which represented the major peak of the immunoprecipitate, showed a strong binding to the hydrophobic matrix. Immunoelectrophoretic quantitation of glycocalicin present in the aqueous media demonstrated that the presence of EDTA, N-ethylmaleimide and iodoacetamide during lysis of platelets significantly reduced the solubilization of glycocalicin. At the same concentrations these inhibitors strongly inhibited the calcium-activated protease of platelet sonicates. Sialic acid determination after acid hydrolysis of aliquots from the soluble fractions showed that their content of sialic acid was considerably higher when lysis was performed in the absence, rather than in the presence, of EDTA and that glycocalicin contributes significantly to the total platelet sialic acid.