Kress B C, Weinreb R N, Pinney E, Miller A L
Department of Neurosciences, University of California, San Diego, La Jolla 92093.
Exp Eye Res. 1991 Dec;53(6):703-7. doi: 10.1016/0014-4835(91)90104-m.
Non-ionic detergent-soluble and detergent-resistant cynomolgus monkey trabecular cell surface glycoconjugates have been identified using three radiolabeling procedures and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The cell surface glycoconjugates were radiolabeled with tritiated sodium borohydride after treatment with: (1) neuraminidase/galactose oxidase; (2) galactose oxidase; or (3) sodium metaperiodate. The non-ionic detergent soluble components were removed by treatment of the monolayer cell culture with 1% Triton X-100 in hypotonic buffer. The radiolabeled trabecular cell surface glycoconjugates in the Triton X-100 soluble and insoluble fractions were resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis under reducing conditions and visualized by fluorography. Treatment of the monkey trabecular cells with neuraminidase/galactose oxidase/tritiated sodium borohydride resulted in the radiolabeling of five components ranging in apparent molecular weight from 128,000 to 39,000 in the Triton X-100 soluble fractions, while 12 radiolabeled glycoconjugates with apparent molecular weights ranging from 240,000 to 26,000 were resolved in the Triton X-100 resistant fraction.
使用三种放射性标记程序以及在十二烷基硫酸钠存在下的聚丙烯酰胺凝胶电泳,已鉴定出食蟹猴小梁细胞表面的非离子去污剂可溶性和去污剂抗性糖缀合物。在用以下物质处理后,细胞表面糖缀合物用氚化硼氢化钠进行放射性标记:(1)神经氨酸酶/半乳糖氧化酶;(2)半乳糖氧化酶;或(3)偏高碘酸钠。通过在低渗缓冲液中用1% Triton X-100处理单层细胞培养物来去除非离子去污剂可溶性成分。在还原条件下,通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳分离Triton X-100可溶性和不溶性部分中的放射性标记小梁细胞表面糖缀合物,并通过荧光自显影进行可视化。用神经氨酸酶/半乳糖氧化酶/氚化硼氢化钠处理猴小梁细胞后,在Triton X-100可溶性部分中产生了5种表观分子量在128,000至39,000之间的放射性标记成分,而在Triton X-100抗性部分中分离出了12种表观分子量在240,000至26,000之间的放射性标记糖缀合物。