Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.
Pre-steady-state charge translocation in NaK-ATPase from eel electric organ.
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DOI：
10.1085/jgp.102.4.631
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发表时间：
1993-10
期刊：
影响因子：
--
通讯作者：
Froehlich JP
中科院分区：
文献类型：
--
作者：
Fendler K;Jaruschewski S;Hobbs A;Albers W;Froehlich JP
时间分辨测量的电荷转移和磷酸化动力学在前稳态的NaK-ATP酶反应周期。从Electrophorus electricus的电器官制备的含NaK-ATP酶的微粒体被吸附到平面脂质双层上用于电荷转移的调查，而快速酸淬灭被用于研究伴随的参与磷酸酶形成的酶促部分反应。为了便于比较这些数据，将条件相对于pH（6.2）、离子组成和温度（24 ° C）标准化。在各种条件下，由酶产生的电流的不同阶段进行了分析，并与磷酸酶形成的动力学进行了比较。最慢的时间常数（τ 3（-1）约为8 s-1）与吸附的膜碎片的电容耦合对电信号的影响有关。与电信号衰减相关联的弛豫时间（τ 2（-1）= 10-70 s-1）取决于ATP和笼状ATP浓度。它被分配给ATP和笼状ATP的结合和交换反应。提出了一个动力学模型，解释了在不同的ATP和笼ATP浓度的弛豫时间的行为。快速混合技术的控制测量证实了这一分配。电信号的上升阶段进行了分析与动力学模型的基础上凝聚的Albers-Post循环。结合从快速混合研究中获得的动力学信息，分析表明，电中性ATP释放，ATP和笼状ATP结合，交换和磷酸化之后是快速的产电E1 P-> E2 P转换。在24 ℃和pH6.2时，鳗鱼电器器官NaK-ATPase中E1 P → E2 P转变的速率常数≥ 1,000 s- 1。
Time-resolved measurements of charge translocation and phosphorylation kinetics during the pre-steady state of the NaK-ATPase reaction cycle are presented. NaK-ATPase-containing microsomes prepared from the electric organ of Electrophorus electricus were adsorbed to planar lipid bilayers for investigation of charge translocation, while rapid acid quenching was used to study the concomitant enzymatic partial reactions involved in phosphoenzyme formation. To facilitate comparison of these data, conditions were standardized with respect to pH (6.2), ionic composition, and temperature (24 degrees C). The different phases of the current generated by the enzyme are analyzed under various conditions and compared with the kinetics of phosphoenzyme formation. The slowest time constant (tau 3(-1) approximately 8 s-1) is related to the influence of the capacitive coupling of the adsorbed membrane fragments on the electrical signal. The relaxation time associated with the decaying phase of the electrical signal (tau 2(-1) = 10-70 s-1) depends on ATP and caged ATP concentration. It is assigned to the ATP and caged ATP binding and exchange reaction. A kinetic model is proposed that explains the behavior of the relaxation time at different ATP and caged ATP concentrations. Control measurements with the rapid mixing technique confirm this assignment. The rising phase of the electrical signal was analyzed with a kinetic model based on a condensed Albers-Post cycle. Together with kinetic information obtained from rapid mixing studies, the analysis suggests that electroneutral ATP release, ATP and caged ATP binding, and exchange and phosphorylation are followed by a fast electrogenic E1P-->E2P transition. At 24 degrees C and pH 6.2, the rate constant for the E1P-- >E2P transition in NaK-ATPase from eel electric organ is > or = 1,000 s- 1.