Brodsky J L, Guidotti G
Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02138.
Am J Physiol. 1990 May;258(5 Pt 1):C803-11. doi: 10.1152/ajpcell.1990.258.5.C803.
The sodium affinities for the two forms of the Na(+)-K(+)-ATPase in brain were characterized. To mimic physiological conditions, synaptosomes, which are pinched off presynaptic nerve termini, were used. Examination of the pump in vitro was performed by preparing synaptic plasma membranes (SPMs). It was first shown that synaptosomes contain the two forms of the Na(+)-K(+)-ATPase, alpha 1 and alpha 2, and that these forms have markedly different affinities for the inhibitory cardiac glycoside ouabain. The apparent dissociation constant (K0.5) of alpha 1 for sodium changed from 12 to 9 mM when going from synaptosomes to membranes. For alpha 2, however, a shift from 36 to 12.5 mM was evident. The conclusion is that in vivo alpha 2 exists as a low sodium affinity species but can be altered to a high-affinity form simply by vesicle disruption. By comparison, the Na(+)-K(+)-ATPase from the mouse fibroblast cell line, 3T3-F442A cells, expressed only the alpha 1-isozyme, as shown by immunoblotting and by measurement of its ouabain and sodium affinities. The physiological relevance of these observations is also presented.
对大脑中两种形式的钠钾ATP酶的钠亲和力进行了表征。为模拟生理条件，使用了从突触前神经末梢分离出的突触体。通过制备突触质膜（SPM）对体外的泵进行检测。首先表明，突触体含有两种形式的钠钾ATP酶，α1和α2，并且这些形式对抑制性强心苷哇巴因具有明显不同的亲和力。从突触体到膜时，α1对钠的表观解离常数（K0.5）从12 mM变为9 mM。然而，对于α2，明显从36 mM转变为12.5 mM。结论是，在体内α2以低钠亲和力形式存在，但仅通过囊泡破坏就可改变为高亲和力形式。相比之下，如免疫印迹以及对其哇巴因和钠亲和力的测量所示，来自小鼠成纤维细胞系3T3-F442A细胞的钠钾ATP酶仅表达α1同工酶。还介绍了这些观察结果的生理相关性。
