Institute for Photobiology of Cells and Organelles, Brandeis University, 02254, Waltham, MA, USA.
Planta. 1985 Jul;165(1):12-22. doi: 10.1007/BF00392206.
Chloroplasts observed, by electron microscopy, to be intact and uncontaminated, with high rates of light-dependent protein synthesis and CO2 fixation were isolated from cells grown on low-vitamin-B12 medium in the light or from cells grown in the same medium in the dark and then exposed to light for 36 h. Both types of chloroplasts were active but less variability was encountered with developing chloroplasts from 36-h cells. The 36-h chloroplasts showed good light-dependent incorporation of 5-amino-levulinic acid (ALA) or L-glutamic acid into chlorophyll (Chl) a which was linear for approx. 1 h. The specific activity of the Chl a remained the same after conversion to pheophytin a, methylpheophorbide a or pyromethylpheophorbide a and rechromatography, indicating that the label was in the tetrapyrrole. Incorporation of ALA was inhibited by levulinic acid, and by chloramphenicol and other inhibitors of translation of 70S-type chloroplast ribosomes at concentrations which did not appreciably inhibit photosynthesis but which blocked plastid protein synthesis nearly completely. Cycloheximide, an inhibitor of translation on 87S cytoplasmic ribosomes of Euglena, was without effect. The 70S inhibitors did not block uptake of labeled ALA. Although labeled glycine was taken up by the plastids, no incorporation into Chl a was observed. Thus the developing chloroplasts appear to contain all of the enzymatic machinery necessary to convert glutamic acid to Chl via the C5 pathway of ALA formation but the Shemin pathway from succinyl coenzyme A and glycine to ALA appears to be absent. The requirement for plastid protein synthesis concomitant with Chl synthesis indicates a regulatory interaction and also indicates that at least one protein influencing Chl synthesis is synthesized on 70S-type plastid ribosomes and is subject to metabolic turnover.
用电子显微镜观察到，从在低维生素 B12 培养基中生长的细胞中分离出来的叶绿体完整且未被污染，具有高比率的依赖光的蛋白质合成和 CO2 固定；或者从在相同培养基中黑暗中生长然后暴露在光下 36 小时的细胞中分离出来的叶绿体也是完整且未被污染的。这两种类型的叶绿体都有活性，但从 36 小时细胞中发育的叶绿体中遇到的可变性较小。36 小时的叶绿体显示出良好的依赖光的 5-氨基-乙酰丙酸（ALA）或 L-谷氨酸掺入叶绿素（Chl）a 的能力，约 1 小时呈线性。在转化为脱镁叶绿酸 a、甲酰基叶绿素 a 或焦甲酰基叶绿素 a 并重新进行色谱分析后，Chl a 的比活性保持不变，表明标记位于四吡咯中。ALA 的掺入被乙酰丙酸、氯霉素和其他翻译 70S 型叶绿体核糖体的抑制剂抑制，这些抑制剂的浓度不会显著抑制光合作用，但几乎完全阻断质体蛋白合成。Euglena 的 87S 细胞质核糖体翻译抑制剂环己酰亚胺没有效果。70S 抑制剂不阻断标记 ALA 的摄取。尽管标记的甘氨酸被质体摄取，但未观察到掺入 Chl a。因此，发育中的叶绿体似乎包含将谷氨酸转化为 Chl 的所有酶促机制，通过 ALA 形成的 C5 途径，但似乎不存在从琥珀酰辅酶 A 和甘氨酸到 ALA 的 Shemin 途径。与 Chl 合成同时需要质体蛋白合成表明存在调节相互作用，并且还表明至少一种影响 Chl 合成的蛋白质是在 70S 型质体核糖体上合成的，并受到代谢周转的影响。
