Department of Biochemistry, Michigan State University, East Lansing, Michigan 48824.
Plant Physiol. 1988 Nov;88(3):543-6. doi: 10.1104/pp.88.3.543.
Cells of Dunaliella tertiolecta from the log phase of growth were broken by rapid extrusion at low pressure through a Yeda press and the chloroplasts were isolated by centrifugation through a Percoll gradient. Osmolarity of the growth media, the suspending media, and the Percoll gradient was kept identical to minimize change in chloroplast volume and mitochondrial entrapment. The isolated intact chloroplasts were obtained in a 30 to 50% yield based on chlorophyll and were stable to washing with buffered medium. Isolated chloroplast yield and purity was dependent on cell culture condition; a cycle of 16 hours light and 8 hours dark with continuous high CO(2) was optimum. Isolated chloroplasts were about 90% intact by microscopic examination, ferricyanide-dependent O(2) evolution, and the distribution of four stromal enzymes. Enzymes associated with glycolate metabolism were not in the chloroplast fraction. The isolated chloroplasts with 10 millimolar bicarbonate evolved 24 micromoles of O(2) and fixed 21 micromoles of CO(2) per hour per milligram of chlorophyll, which rates were about one-third of those by whole cells. The inhibition of oxygen evolution by 10 millimolar phosphate was reversed by P-glycerate. Whole chloroplasts were also isolated from cells adapted to low CO(2) in air for 24 hours. On low CO(2) the cells excreted more gelatinous material, which had to be removed with additional washing of the cells, before it was possible to obtain good chloroplast preparations.
从对数生长期的杜氏盐藻细胞中通过快速低压挤压通过 Yeda 压碎机破碎，然后通过 Percoll 梯度离心分离叶绿体。生长培养基、悬浮培养基和 Percoll 梯度的渗透压保持一致，以最大程度地减少叶绿体体积和线粒体捕获的变化。根据叶绿素的含量，以 30%至 50%的产量获得完整的分离叶绿体，并且用缓冲介质洗涤稳定。分离的叶绿体的产量和纯度取决于细胞培养条件；16 小时光照和 8 小时黑暗，持续高 CO2 的循环是最佳的。通过显微镜检查、铁氰化物依赖性 O2 演化和四个基质酶的分布，发现分离的叶绿体约有 90%完整。与糖酵解代谢相关的酶不在叶绿体部分。用 10 毫摩尔碳酸氢盐进行演化，分离的叶绿体每小时每毫克叶绿素可演化 24 微摩尔的 O2 和固定 21 微摩尔的 CO2，其速率约为完整细胞的三分之一。10 毫摩尔磷酸盐对氧演化的抑制可被 P-甘油酸逆转。还从适应空气低 CO2 24 小时的细胞中分离出完整的叶绿体。在低 CO2 下，细胞分泌出更多的凝胶状物质，在获得良好的叶绿体制剂之前，必须用细胞的额外洗涤去除。
