DiPolo R, Berberián G, Delgado D, Rojas H, Beaugé L
Center de Biofisica y Bioquímica, IVIC, Caracas, Venezuela.
FEBS Lett. 1997 Jan 13;401(1):6-10. doi: 10.1016/s0014-5793(96)01416-0.
The Na/Ca exchange is a highly regulated transport mechanism in which MgATP, a powerful modulatory intracellular substrate, has important implications for its function. As occurs with some preparations, in squid axons, nucleotide regulation is lost after membrane vesicle isolation. This has been a significant obstacle in the biochemical characterization of the MgATP effect. An important clue in solving this long-standing puzzle is presented in this work by showing that prolonged intracellular dialysis of squid axons produces a complete run down of the MgATP effect. Here we report that a soluble cytoplasmic factor isolated from fresh squid axoplasm and brain reconstitutes the MgATP stimulation of the Na-gradient-dependent 45Ca uptake in squid optic nerve membrane vesicles. Partial purification of this factor uncovers the presence of a novel 13 kDa soluble cytoplasmic protein (SCPr) which, when microinjected in ATP de-regulated dialyzed squid axons, completely restores the MgATP stimulation of Na(o)-dependent Ca efflux. We propose that in the squid preparation this SCPr constitutes the link between the nucleotide and target effector: the Na/Ca exchanger itself, or other plasma membrane structures which may secondarily interact with the exchanger.
钠/钙交换是一种高度受调控的转运机制，其中MgATP（一种强大的细胞内调节底物）对其功能具有重要影响。正如在一些标本中所发生的那样，在枪乌贼轴突中，膜囊泡分离后核苷酸调节作用丧失。这一直是MgATP效应生化特性研究中的一个重大障碍。这项工作提供了一个解决这一长期难题的重要线索，即通过表明对枪乌贼轴突进行长时间的细胞内透析会导致MgATP效应完全消失。我们在此报告，从新鲜枪乌贼轴浆和脑组织中分离出的一种可溶性细胞质因子可重建枪乌贼视神经膜囊泡中MgATP对钠梯度依赖性45Ca摄取的刺激作用。对该因子的部分纯化揭示了一种新的13 kDa可溶性细胞质蛋白（SCPr）的存在，当将其显微注射到ATP调节失控的透析枪乌贼轴突中时，可完全恢复MgATP对钠依赖性钙外流的刺激作用。我们提出，在枪乌贼标本中，这种SCPr构成了核苷酸与靶效应器（钠/钙交换体本身或可能与交换体发生二次相互作用的其他质膜结构）之间的联系。
