Dunahay T G, Staehelin L A
Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, Colorado 80309.
Plant Physiol. 1985 Jul;78(3):606-13. doi: 10.1104/pp.78.3.606.
We have used the nonionic detergent octyl-beta-d-glucopyranoside in combination with sodium dodecyl sulfate to isolate two novel Photosystem I (PSI) complexes from spinach (Spinacea oleracea L.) thylakoid membranes. These complexes have been characterized as to their spectral properties, content of PSI reaction center chlorophyll P(700), and protein composition. PSI-B, purified from solubilized membranes by sucrose density gradient centrifugation, is a putative native PSI complex. PSI-B contains four polypeptides between 21 and 25 kilodaltons in addition to the components of the PSI antenna complex (LHCI); three of these polypeptides have not previously been associated with PSI. A second complex, CPI(*), is purified from octyl glucoside/sodium dodecyl sulfate solubilized thylakoids by two cycles of preparative gel electrophoresis under mildly denaturing conditions. Electrophoresis under these conditions releases a discrete set of polypeptides from PSI producing a complex composed only of the PSI reaction center and the LHCI antenna.In addition, the PSI reaction center complex CPI isolated from preparative gels and PSI-B were reconstituted into lecithin liposomes for structural analysis using freeze-fracture electron microscopy. The results suggest that the native PSI complex produces 12- to 13-nanometer particles, while the PSI reaction center, depleted of LHCI and peripheral proteins, produces particles with an average diameter of 10 nanometers.
我们使用非离子去污剂辛基-β-D-吡喃葡萄糖苷与十二烷基硫酸钠相结合，从菠菜（Spinacea oleracea L.）类囊体膜中分离出两种新型光系统I（PSI）复合物。已对这些复合物的光谱特性、PSI反应中心叶绿素P(700)的含量以及蛋白质组成进行了表征。通过蔗糖密度梯度离心从溶解的膜中纯化得到的PSI-B是一种假定的天然PSI复合物。除了PSI天线复合物（LHCI）的成分外，PSI-B还包含四种分子量在21至25千道尔顿之间的多肽；其中三种多肽以前未与PSI相关联。第二种复合物CPI(*)是在温和变性条件下通过两轮制备性凝胶电泳从辛基葡萄糖苷/十二烷基硫酸钠溶解的类囊体中纯化得到的。在这些条件下进行电泳会从PSI中释放出一组离散的多肽，从而产生仅由PSI反应中心和LHCI天线组成的复合物。此外，从制备性凝胶中分离出的PSI反应中心复合物CPI和PSI-B被重构到卵磷脂脂质体中，以便使用冷冻断裂电子显微镜进行结构分析。结果表明，天然PSI复合物产生12至13纳米的颗粒，而去除LHCI和外周蛋白的PSI反应中心产生平均直径为10纳米的颗粒。
