Agalakova N I, Lapin A V, Gusev G P
Sechenov Institute of Evolutionary Physiology and Biochemistry, St. Petersburg, Russia.
J Comp Physiol B. 1997 Nov;167(8):576-81. doi: 10.1007/s003600050112.
This study was undertaken to evaluate the effects of various metabolic blockers on the Na-K-pump activity and ATP content of frog erythrocytes. To eliminate K-Cl cotransport, the frog erythrocytes were incubated in nitrate media at 20 degrees C. Incubation of the red cells in a glucose-free medium for 2 h had no effect on cell ATP content and K+ influx measured as 86Rb uptake for 60 min. The Na(+)-K(+)-pump activity was also unchanged in the frog erythrocytes incubated in a glucose-free medium containing 10 mM 2-deoxy-D-glucose or adenosine. Unexpectedly, the treatment of red cells with 1-2 mM glycolytic blocker iodoacetate produced a 2-fold increase in the ouabain-sensitive K+ influx. The cell ATP content declined by 9.4% after 2 h of cell incubation with iodoacetate. Incubation of the red cells for 90 min in the presence of 2 mM cyanide, 0.01 mM antimycin A or 5 mM azide resulted in a significant reduction in K+ influx by about 50%, 45% and 32%, respectively. The cell ATP content diminished over 60 min and 120 min of cell incubation with 2 mM cyanide by 15.6% and 31.7% of control levels, respectively. In time-course experiments, a 50% reduction in the K+ influx was observed when the frog erythrocytes were incubated for only 30 min in the presence of 2 mM cyanide. In contrast, 0.01-0.10 mM rotenone, a site I inhibitor, and 0.01 mM carbonyl cyanide m-chlorophenylhydrazone, an uncoupler of oxidative phosphorylation were without effect on K+ influx into frog erythrocytes. These results indicate that about one-half of the Na(+)-K(+)-pump activity in frog erythrocytes is tightly functionally coupled to cytochromes via a separate "membrane-associated" ATP pool.
本研究旨在评估各种代谢阻滞剂对青蛙红细胞钠钾泵活性和ATP含量的影响。为消除钾氯共转运，将青蛙红细胞置于20摄氏度的硝酸盐培养基中孵育。在无葡萄糖培养基中孵育红细胞2小时，对细胞ATP含量和以60分钟内86Rb摄取量衡量的钾离子流入量没有影响。在含有10 mM 2-脱氧-D-葡萄糖或腺苷的无葡萄糖培养基中孵育的青蛙红细胞，其钠钾泵活性也未改变。出乎意料的是，用1-2 mM糖酵解阻滞剂碘乙酸处理红细胞后，哇巴因敏感的钾离子流入量增加了两倍。用碘乙酸孵育细胞2小时后，细胞ATP含量下降了9.4%。在2 mM氰化物、0.01 mM抗霉素A或5 mM叠氮化物存在的情况下，将红细胞孵育90分钟，钾离子流入量分别显著减少约50%、45%和32%。用2 mM氰化物孵育细胞60分钟和120分钟后，细胞ATP含量分别降至对照水平的15.6%和31.7%。在时间进程实验中，当青蛙红细胞在2 mM氰化物存在的情况下仅孵育30分钟时，观察到钾离子流入量减少了50%。相比之下，0.01-0.10 mM鱼藤酮（一种位点I抑制剂）和0.01 mM羰基氰化物间氯苯腙（一种氧化磷酸化解偶联剂）对青蛙红细胞钾离子流入没有影响。这些结果表明，青蛙红细胞中约一半的钠钾泵活性通过一个独立的“膜相关”ATP池与细胞色素在功能上紧密耦合。
