Na+,K+-ATPase interaction with a brain endogenous inhibitor (endobain E)

Na+,K+-ATPase activity of rat brain synaptosomal membranes was evaluated in the presence of an inhibitory fraction II-E (termed endobain E), isolated by gel filtration and anionic exchange HPLC of a rat brain soluble fraction . We studied endobain E aging, analyzed its inhibitory potency in the absen ce or presence of ouabain as well as its ability to block high affinity [H- 3]ouabain binding to cerebral cortex membranes. Similar loss of endobain E activity was observed when samples were stored either dried or in solution. Endobain E fraction inhibited synaptosomal membrane Na+,K+-ATPase activity in a concentration-dependent manner and the slope of the corresponding cur ve strongly resembled that of ouabain. Assays performed in the presence of endobain E and ouabain indicated that the inhibitory effect was additive or less than additive, depending on their respective concentrations during pr eincubation and/or incubation. High affinity [H-3]ouabain binding to cerebr al cortex membranes proved concentration-dependent from 0.10 to 0.50 mg pro tein per mi; binding inhibition by endobain E was independent of protein co ncentration within the above range. [H-3]ouabain binding inhibition by endo bain E was concentration-dependent over a 10-fold range, an effect similar to that found for Na+,K+-ATPase inhibition. The extent of endobain E effect on Na+,K+-ATPase inhibition was much higher (90-100%) than that on [3H]oua bain binding blockade (50%). Findings suggest some type of interaction betw een endobain E and ouabain inhibitory mechanisms and favour the view that t he former behaves as an endogenous ouabain. (C) 1998 Elsevier Science Ltd. All rights reserved.