Novel microsomal anion-sensitive Mg 2+-ATPase activity in rat brain

Abstract

Ethacrynic acid (EA) highly sensitive Mg 2+-ATPase activity was demonstrated in rat brain microsomes. Marker enzyme studies suggested that the EA highly sensitive Mg 2+-ATPase activity originated mainly from plasma membranes, and possibly from synaptic vesicles. Oligomycin did not affect the EA highly sensitive Mg 2+-ATPase activity. Sulfhydryl reagents, such as N-ethylmaleimide and 5,5′-dithiobis-(2-nitrobenzoic acid), and anion transport inhibitors, such as 4-acetamide-4′-isothiocyano-stilbene-2,2′-disulfonic acid, 4,4′-diisothiocyano-stilbene-2,2′-disulfonic acid and 2,4-dinitro-1-fluorobenzene, completely inhibited the EA highly sensitive Mg 2+-ATPase activity with apparent K i values at 5, 5, 8, 8 and 10 μM respectively. Treatment of microsomes with ethylenediaminetetraacetic acid and ammonium sulfate increased the EA highly sensitive Mg 2+ and Na +,K +-ATPase activities, but not EA less sensitive Mg 2+- or HCO 3-ATPase activity, 2- to 3-fold that in crude microsomes. Relative substrate specificities of ATP ⪢ GTP > ITP > UTP, CTP, a K m for ATP at 0.77 mM, and an optimal pH at pH 7.4 were observed. Among the anions tested (Cl −, Br −, F −, HCO 3 −, I −, SCN −, NO 3 −), EA highly sensitive Mg 2+-ATPase activity was stimulated significantly by Cl − and reduced by NO 3 −. These data suggest that a novel, plasma membrane-located and anion-sensitive Mg 2+-ATPase activity exists in the brain.