Institut für Allgemeine Botanik und Botanischer Garten, Universität Hamburg, Ohnhorststrasse, 18, D-2000, Hamburg 52, Federal Republic of Germany.
Planta. 1985 Jun;164(3):379-89. doi: 10.1007/BF00402950.
A procedure has been developed to isolate and analyse the cell-wall glycoproteins of Chlamydomonas reinhardii. Under appropriate conditions, cell-wall glycoproteins can be quantitatively extracted from intact cells by aqueous LiCl. Although proteins and glycoproteins, which are presumably not related to the cell wall, are coextracted with the cell-wall subunits, these components can be readily identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis as demonstrated by comparative analysis of LiCl-extracts from wild-type cells and the cell-wall-deficient mutant CW-15. Apart from the high-molecular-weight cell-wall components, two glycoproteins with apparent molecular weights (Mrs) of 36000 and 66000 were found to be present in LiCl-extracts of wild-type cells but absent in LiCl-extracts from the cell-wall-less mutant. Pulse-labeling experiments with [(3)H]proline and [(35)S]methionine revealed that the LiCl-extracts contained - in addition to the well-known cell-wall subunits - proteins of lower molecular weight, which are also preferentially labeled with [(3)H]proline. Protein components with Mrs of 68000, 44000, 36000, 26000 and 22000 were found to be more strongly labeled with [(3)H]proline than with [(35)S]methionine, whereas protein components with Mrs of 57000 and 52000 were more prominent after labeling with [(35)S]methionine. The portion of cell-wall subunits within the total amount of proteins extracted by LiCl was calculated to be at least 10% on the basis of the amount of hydroxyproline. Self-assembly of cell walls could be demonstrated after dialysis against water of a mixture of crude LiCl-extract and purified, insoluble, inner wall layers. Cell-wall glycoproteins could be enriched by gel exclusion chromatography of crude LiCl-extracts on Sepharose CL-4B columns equilibrated with 1 mol l(-1) LiCl.
已经开发出一种从莱茵衣藻中分离和分析细胞壁糖蛋白的方法。在适当的条件下，细胞壁糖蛋白可以通过水相 LiCl 从完整细胞中定量提取。尽管与细胞壁没有关系的蛋白质和糖蛋白也与细胞壁亚基一起被共提取，但这些成分可以通过 SDS-聚丙烯酰胺凝胶电泳很容易地被识别出来，这可以通过比较分析来自野生型细胞和细胞壁缺陷突变体 CW-15 的 LiCl 提取物来证明。除了高分子量的细胞壁成分外，还发现两种糖蛋白在 LiCl 提取物中的表观分子量（Mr）为 36000 和 66000，存在于野生型细胞的 LiCl 提取物中，但不存在于无细胞壁的突变体的 LiCl 提取物中。用 [(3)H]脯氨酸和 [(35)S]甲硫氨酸进行脉冲标记实验表明，LiCl 提取物除了已知的细胞壁亚基外，还含有低分子量的蛋白质，这些蛋白质也优先被 [(3)H]脯氨酸标记。Mr 为 68000、44000、36000、26000 和 22000 的蛋白质成分被 [(3)H]脯氨酸标记的强度比 [(35)S]甲硫氨酸标记的强度高，而 Mr 为 57000 和 52000 的蛋白质成分在用 [(35)S]甲硫氨酸标记后更为明显。根据羟基脯氨酸的量，用 LiCl 提取的总蛋白质中细胞壁亚基的部分至少为 10%。在水透析后，可以证明 crude LiCl-extract 和纯化的、不可溶的内壁层的混合物的自组装。可以通过将 crude LiCl-extract 在 Sepharose CL-4B 柱上进行凝胶排阻层析，用 1 mol l(-1) LiCl 平衡，从而富集细胞壁糖蛋白。
