Blum H, Balschi J A, Johnson R G
Department of Medicine, University of Pennsylvania Medical Center, Philadelphia 19104.
J Biol Chem. 1991 Jun 5;266(16):10254-9.
Physiological control of the plasma membrane sodium pump, (Na+,K+)-ATPase, is essential for proper function of eukaryotic cells. In the electric organ of the elasmobranch Narcine brasiliensis, the normal demands placed upon the pump during the process of generation of electrical currents call for large and rapid changes in activity of this enzyme, making this a good model for the study of its cellular regulation. 31P NMR spectroscopic techniques were used to study metabolic regulation of membrane pump function in resting and stimulated electric organ and in skeletal muscle of the live, intact N. brasiliensis. Because the ATP synthetic abilities of the electric organ by glycolysis or oxidative phosphorylation are extremely limited, depletion of phosphocreatinine (PCr) could be used to determine the activity of the (Na+,K+)-ATPase after the electric organ was stimulated to discharge, and to measure the net flux from PCr to ATP through the creatine phosphokinase (CPK) reaction in the electric organ. Saturation transfer, an NMR technique which measures exchange rates, was applied to determine the unidirectional flux in the forward direction through the same reaction in the electric organ and in skeletal muscle as a control. The pseudo first-order rate constant kf for the CPK reaction at 24 degrees C in resting electric organ was 0.000 +/- 0.002 s-1 (n = 10) and in skeletal muscle was 0.08 +/- 0.03 s-1 (n = 3). The results demonstrate that in resting electric organ, which is well supplied with CPK, there was no measurable flux through this reaction, although CPK when extracted is highly active. Measured and calculated levels of all substrates for the creatine kinase reaction in the electric organ are similar to those in unstimulated skeletal muscle, where the creatine phosphokinase reaction rates are high in vivo. In contrast to the resting electric organ, during stimulation of the electric organ the measured net rate constant was greater than 0.08 s-1. In addition, as shown by lack of PCr depletion, there was virtually no net turnover of ATP in the resting organ compared to the stimulated organ. The marked difference in the (Na+,K+)-ATPase activity in the resting and activated electric organ confirmed earlier results (Blum, H., Nioka, S., and Johnson, R. G., Jr. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 1247-1251). Together, these results suggest that there is a novel method of coordinate regulation of cellular enzymes of great sensitivity and rapidity.
质膜钠泵（Na⁺，K⁺）-ATP酶的生理调控对于真核细胞的正常功能至关重要。在巴西电鳐的电器官中，在电流产生过程中对该泵的正常需求要求这种酶的活性发生大幅度快速变化，这使其成为研究其细胞调控的良好模型。利用³¹P NMR光谱技术研究了完整活体巴西电鳐的静息和受刺激的电器官以及骨骼肌中膜泵功能的代谢调控。由于通过糖酵解或氧化磷酸化产生ATP的能力极其有限，磷酸肌酸（PCr）的消耗可用于确定电器官受刺激放电后（Na⁺，K⁺）-ATP酶的活性，并测量通过电器官中肌酸磷酸激酶（CPK）反应从PCr到ATP的净通量。饱和转移是一种测量交换率的NMR技术，用于确定通过电器官和作为对照的骨骼肌中相同反应正向的单向通量。静息电器官在24℃时CPK反应的伪一级速率常数kf为0.000±0.002 s⁻¹（n = 10），骨骼肌中为0.08±0.03 s⁻¹（n = ３）。结果表明，在静息的、CPK供应充足的电器官中，尽管提取的CPK具有高活性，但该反应没有可测量的通量。电器官中肌酸激酶反应所有底物的测量值和计算值与未受刺激的骨骼肌中的相似，在未受刺激的骨骼肌中肌酸磷酸激酶反应速率在体内较高。与静息电器官相反，在电器官受刺激期间，测量的净速率常数大于0.08 s⁻¹。此外，如PCr未消耗所示，与受刺激的器官相比，静息器官中实际上没有ATP的净周转。静息和激活的电器官中（Na⁺，K⁺）-ATP酶活性的显著差异证实了早期结果（Blum，H.，Nioka，S.，和Johnson，R.G.，Jr.（199０）Proc.Natl.Acad.Sci.U.S.A.87，1247 - 1251）。总之，这些结果表明存在一种对细胞酶进行协调调控的新方法，具有高度的敏感性和快速性。
