Kreimer G, Brohsonn U, Melkonian M
Botanisches Institut, Lehrstuhl I, Universität Köln, Bundesrepublik Deutschland.
Eur J Cell Biol. 1991 Aug;55(2):318-27.
We report on the isolation and purification of structurally intact eyespot apparatuses from the naked, biflagellate green alga Spermatozopsis similis. Two eyespot-enriched fractions, separated by sucrose gradient centrifugation, retained the typical reflective properties of eyespots in situ as demonstrated by reflection confocal laser scanning microscopy. Ultrastructurally, both fractions contained eyespot plates consisting of a single layer of lipid globules. Structurally intact eyespot apparatuses, including patches of plasma membrane and chloroplast envelope overlying the eyespot plate and a single thylakoid subtending the eyespot plate, were particularly enriched in one of the two fractions (fraction 2a). Measurement of several marker enzymes and chlorophyll content (less than 0.001% of total) established the absence of most other cell organelles from the eyespot fractions. The absorption spectra of the two fractions were dominated by carotenoids with an additional shoulder at 540 nm. Following extraction with organic solvents and sodium dodecyl sulfate polyacrylamide gel electrophoresis, several proteins were found to be considerably enriched in the two fractions. In addition to several proteins in the high Mr range, at least 4 polypeptides of 35, 29, 23, and 20 kDa are selectively enriched in fraction 2a with the 29 and 20 kDa proteins being the most prominent. The presence of glycoproteins in fraction 2a was demonstrated by binding of the mannose-specific lectin Galanthus nivalis agglutinin to several high molecular weight polypeptides. In addition, a hydrophobic component with abnormal electrophoretic mobility that reacts strongly with periodic acid-Schiff and thymol/sulfuric acid was prominent in both fractions. Mass isolation and purification of the intact phototactic apparatus of a flagellate green alga now greatly facilitates the biochemical and molecular characterization of the signal transduction chain involved in green algal phototaxis.
我们报道了从裸露的双鞭毛绿藻精子藻中分离和纯化结构完整的眼点装置的过程。通过蔗糖梯度离心分离得到的两个富含眼点的组分，经反射共聚焦激光扫描显微镜证实，保留了原位眼点的典型反射特性。超微结构上，两个组分均含有由单层脂质球组成的眼点板。结构完整的眼点装置，包括覆盖眼点板的质膜和叶绿体被膜斑块以及位于眼点板下方的单个类囊体，在两个组分之一（组分2a）中尤为富集。对几种标记酶和叶绿素含量（占总量不到0.001%）的测定表明，眼点组分中不存在大多数其他细胞器。两个组分的吸收光谱以类胡萝卜素为主，在540 nm处有一个附加峰。用有机溶剂和十二烷基硫酸钠聚丙烯酰胺凝胶电泳提取后，发现几种蛋白质在两个组分中大量富集。除了几种高分子量范围内的蛋白质外，至少有4种35、29、23和20 kDa的多肽在组分2a中选择性富集，其中29和20 kDa的蛋白质最为突出。通过甘露糖特异性凝集素雪花莲凝集素与几种高分子量多肽的结合，证明了组分2a中存在糖蛋白。此外，一种电泳迁移异常且与高碘酸 - 席夫试剂和百里酚/硫酸反应强烈的疏水成分在两个组分中都很突出。鞭毛绿藻完整趋光装置的大量分离和纯化现在极大地促进了绿藻趋光性中信号转导链的生化和分子特征研究。
