Jenkins C L, Boag S
CSIRO, Division of Plant Industry, GPO Box 1600, Canberra A.C.T. 2601, Australia.
Plant Physiol. 1985 Sep;79(1):84-9. doi: 10.1104/pp.79.1.84.
Bundle sheath chloroplasts have been isolated from Zea mays leaves by a procedure involving enzymic digestion of mechanically prepared strands of bundle sheath cells followed by gentle breakage and filtration. The resulting crude chloroplast preparation was enriched by Percoll density layer centrifugation to yield intact chloroplasts (about 20 micrograms chlorophyll per 10-gram leaf tissue) with high metabolic activities. Based on activities of marker enzymes in the chloroplast and bundle sheath cell extracts, the chloroplasts were essentially free of contamination by other organelles and cytoplasmic material, and were generally about 70% intact. Chlorophyll a/b ratios were high (about 10). With appropriate substrates these chloroplasts displayed high rates of malate decarboxylation, measured as pyruvate formation, and CO(2) assimilation (maximum rates approximately 5 and 3 micromoles per minute per milligram chlorophyll, respectively). These activities were light dependent, linear for at least 20 minutes at 30 degrees C, and displayed highest rates at pH 8.0. High metabolic rates were dependent on addition of an exogenous source of carbon to the photosynthetic carbon reduction cycle (3-phosphoglycerate or dihydroxyacetone phosphate) and a nucleotide (ATP, ADP, or AMP), as well as aspartate. Generally, neither malate decarboxylation nor CO(2) assimilation occurred substantially in the absence of the other activity indicating a close relationship between these processes. Presumably, NADPH required for the photosynthetic carbon reduction cycle is largely supplied during the decarboxylation of malate by NADP-malic enzyme. The results are discussed in relation to the role of bundle sheath chloroplasts in C(4) photosynthesis by species of the NADP-malic enzyme type.
通过一种方法从玉米叶片中分离出了维管束鞘叶绿体，该方法包括对机械制备的维管束鞘细胞条进行酶消化，然后轻轻破碎并过滤。所得的粗叶绿体制剂通过Percoll密度梯度离心进行富集，以产生具有高代谢活性的完整叶绿体（每10克叶片组织约含20微克叶绿素）。根据叶绿体和维管束鞘细胞提取物中标记酶的活性，这些叶绿体基本上没有受到其他细胞器和细胞质物质的污染，并且通常约70%是完整的。叶绿素a/b比值很高（约为10）。在合适的底物存在下，这些叶绿体表现出高的苹果酸脱羧速率（以丙酮酸形成来衡量）和CO₂同化速率（最大速率分别约为每分钟每毫克叶绿素5和3微摩尔）。这些活性依赖于光，在30℃下至少20分钟呈线性，并且在pH 8.0时表现出最高速率。高代谢速率依赖于向光合碳还原循环中添加外源碳源（3-磷酸甘油酸或磷酸二羟丙酮）、一种核苷酸（ATP、ADP或AMP）以及天冬氨酸。一般来说，在没有另一种活性的情况下，苹果酸脱羧和CO₂同化基本上都不会发生，这表明这些过程之间存在密切关系。据推测，光合碳还原循环所需的NADPH在很大程度上是在苹果酸由NADP-苹果酸酶脱羧过程中提供的。讨论了这些结果与NADP-苹果酸酶类型物种的维管束鞘叶绿体在C₄光合作用中的作用的关系。
