CHLAMYDOMONAS-REINHARDTII CELL PREPARATION WITH ALTERED PERMEABILITY TOWARD SUBSTRATES OF ORGANELLAR REACTIONS
CHLAMYDOMONAS-REINHARDTII CELL PREPARATION WITH ALTERED PERMEABILITY TOWARD SUBSTRATES OF ORGANELLAR REACTIONS
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DOI：
10.1073/pnas.78.4.2310
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发表时间：
1981-01-01
期刊：
影响因子：
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通讯作者：
TOGASAKI, RK
中科院分区：
文献类型：
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作者：
BELKNAP, WR;TOGASAKI, RK
在 Yeda 压榨机中低压下破碎莱茵衣藻细胞，产生对 A 类叶绿体和完整线粒体的底物具有高渗透性的制剂（压榨液）。野生型细胞加压液的 CO2 吸收和 O2 光演化的化学计量速率被 10 mM 外源磷酸盐严重抑制，并且这种抑制可以通过添加 3-磷酸甘油酸或二羟基丙酮磷酸盐来逆转。通过使用完整细胞、加压液和超声加压液来研究突变体 F60，该突变体缺乏磷酸核糖激酶活性和 CO2 依赖性 O2 光演化。在加压液中，3-磷酸甘油酸依赖性O2光演化速率较高；对 K3Fe(CN)6 的依赖性较低。对苯醌支持加压液和超声处理加压液中高速率的 O2 释放。暗适应野生型加压酸盐在黑暗中对 O2 的缓慢吸收可以通过添加琥珀酸盐和 ADP 的刺激来增加。添加 KCN 会导致该活性快速但仅部分受到抑制。未压制的制剂吸收暗氧没有表现出类似的反应。所描述的程序开启了对莱茵衣野生型和突变株的 A 类叶绿体进行原位分析的可能性。
C. reinhardtii cells disrupted under low pressure in a Yeda press yielded a preparation (pressate) with high permeability toward substrates for Class A chloroplasts and intact mitochondria. The stoichiometric rates of CO2 uptake and O2 photoevolution by the wild-type cell pressate were severely suppressed by 10 mM exogenous phosphate and this suppression could be reversed by the addition of either 3-phosphoglycerate or dihydroxyacetone phosphate. A mutant F60, which lacks phosphoribulokinase activity and CO2-dependent O2 photoevolution, was studied by using intact cells, pressate and sonicated pressate. In the pressate, the rate of 3-phosphoglycerate-dependent O2 photoevolution was high; that dependent on K3Fe(CN)6 was low. p-Benzoquinone supported high rates of O2 evolution in both the pressate and the sonicated pressate. The slow O2 uptake in the dark by the dark-adapted wild-type pressate could be increased by the addition of succinate and stimulated by ADP. Addition of KCN resulted in rapid but only partial suppression of this activity. Dark O2 uptake by the unpressed preparation did not show similar responses. The procedure described opens the possibility of in situ analysis of Class A chloroplasts from wild-type and mutant strains of C. reinhardtii.