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Novel microsomal anion-sensitive Mg2+-ATPase activity in rat brain
Abstract:
Ethacrynic acid (EA) highly sensitive Mg2+-ATPase activity was demonstrated in rat brain microsomes. Marker enzyme studies suggested that the EA highly sensitive Mg2+-ATPase activity originated mainly from plasma membranes, and possibly from synaptic vesicles. Oligomycin did not affect the EA highly sensitive Mg2+-ATPase activity. Sulfhydryl reagents, such as N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid), and anion transport inhibitors, such as 4-acetamide-4'-isothiocyanostilbene-2,2'-disulfonic acid, 4,4'-diisothiocyano-stilbene-2,2'-disulfonic acid and 2,4-dinitro-1-fluorobenzene, completely inhibited the EA highly sensitive Mg2+-ATPase activity with apparent Ki values at 5, 5, 8, 8 and 10 microM respectively. Treatment of microsomes with ethylenediaminetetraacetic acid and ammonium sulfate increased the EA highly sensitive Mg2+ and Na+,K+-ATPase activities, but not EA less sensitive Mg2+- or HCO3-ATPase activity, 2- to 3-fold that in crude microsomes. Relative substrate specificities of ATP much greater than GTP greater than ITP greater than UTP, CTP, a Km for ATP at 0.77 mM, and an optimal pH at pH 7.4 were observed. Among the anions tested (Cl-, Br-, F-, HCO3-, I-, SCN-, NO3-), EA highly sensitive Mg2+-ATPase activity was stimulated significantly by Cl- and reduced by NO3-. These data suggest that a novel, plasma membrane-located and anion-sensitive Mg2+-ATPase activity exists in the brain.
Insights
Researchers identified a novel ethacrynic acid (EA)-sensitive Mg2+-ATPase in rat brain microsomes, primarily located on plasma membranes. This enzyme is sensitive to sulfhydryl reagents and anions, suggesting a new role in brain function.
Area of Science:
- Neurochemistry
- Enzymology
- Membrane Biology
Background:
- Mg2+-ATPase activity is crucial for neuronal function.
- Previous studies have not fully characterized all Mg2+-ATPase isoforms in the brain.
Purpose of the Study:
- To identify and characterize a novel Mg2+-ATPase activity in rat brain microsomes.
- To determine the cellular localization and biochemical properties of this enzyme.
Main Methods:
- Enzyme activity assays using rat brain microsomes.
- Inhibition studies with sulfhydryl reagents and anion transport inhibitors.
- Substrate specificity and kinetic analysis.
Main Results:
- Demonstrated ethacrynic acid (EA)-sensitive Mg2+-ATPase activity.
- Localized the activity primarily to plasma membranes and possibly synaptic vesicles.
- Showed inhibition by sulfhydryl reagents and specific anion transport inhibitors.
- Observed stimulation by chloride and inhibition by nitrate.
Conclusions:
- A novel, plasma membrane-localized, and anion-sensitive Mg2+-ATPase activity exists in the brain.
- This enzyme may play a role in neuronal ion transport and signaling.