Brain (Na+,K+)-ATPase: biphasic interaction with erythrosin B.
Brain (Na+,K+)-adenosine triphosphatase (EC 3.6.1.3) has both high and low affinity ouabain binding sites. It has been proposed that the high affinity ouabain binding sites characterize a nerve-specific form of the enzyme. Erythrosin B has been reported to inhibit high affinity ouabain binding selectively. The experiments in this paper were carried out in order to characterize the interactions of erythrosin B with (Na+,K+)-ATPase and to examine the specificity of erythrosin B for enzyme with high affinity for ouabain. Inhibition by erythrosin B was biphasic, with a rapid and a slow phase. The rapid phase appeared to be relatively specific for enzyme with high affinity for ouabain, while the slow phase was not. Inhibition by erythrosin B was accelerated by Mg2+ and was retarded by ATP, K+, or Na+ and ATP. Erythrosin B increased apparent affinity of the enzyme for K+ and decreased apparent affinity for Na+ and for ATP. These results indicate that erythrosin B interacts with an ATP site and has effects on cation affinities opposite to those of ATP. Erythrosin B inhibition is proportional to high affinity ouabain binding if brief incubation times and moderate concentrations are used.
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D004923:Erythrosine / Q000494:pharmacology*; D005452:Fluoresceins / Q000494:pharmacology*; D066298:In Vitro Techniques; D007700:Kinetics; D008968:Molecular Conformation; D010042:Ouabain / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*
D005452:Fluoresceins; D010042:Ouabain; D000255:Adenosine Triphosphate; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D004923:Erythrosine