Properties of Cl--Stimulated Mg2+-ATPase Sensitive to Ligands of Inhibitory Receptors in Brain Plasma Membranes of the Bream Abramis brama

Abstract

Properties of Cl−-stimulated Mg2+-ATPase in the brain plasma membranes of the bream Abramis brama L. were studied; this enzyme is composed of “basal” Mg2+-ATPase activity that can be stimulated by 40–80% by Cl− ions (Cl−-ATPase). These anions stimulate the basal Mg2+-ATPase starting with 8 mM concentration, their maximal effect being observed at a concentration of 30–100 mM. The Cl−-ATPase activity was found at a low molarity of HEPES-Tris buffer ( 30 mM). The “basal” Mg2+-ATPase activity was detected in the whole studied pH range (5.5–9.0), with maximum at pH 7.2–7.8 values, whereas optimum to reveal Cl−-ATPase was at high and low H+ concentrations (pH 6.0 and 8.5, respectively). At physiological pH values (7.2–7.5) the Cl−-ATPase activity was not revealed, but was detected after preincubation of the enzyme with 10 µM GABA. The “basal” Mg2+-ATPase, like Cl−-ATPase, hydrolyzed ATP with a maximal rate, while CTP, ITP, and ADP only slightly, and did not hydrolyze GTP and AMP. The Cl−-ATPase activity decreased in the presence of divalent cations in the following order: Mg2+ > Co2+ > Mn2+ = Cd2+ > Al3+ = Cu2+, and it was not found in the presence of Ca2+ and Zn2+. Anions of halogen series activated the “basal” Mg2+-ATPase in the descending order: Cl− > Br− > J− > F−. Among other monovalent anions, HCO3− activated the enzyme, NO3− practically had no effect, and SCN− inhibited its activity. Blockers of Cl− transport (ethacrinic acid, furosemide, and SITS) and GABA-receptor ligands (pentobarbital, diazepam, and picrotoxin) suppressed the enzyme activity. Out of SH-reagents, PCMB inhibited the enzyme, while NEM did not affect it. The H+-ATPase blocker oligomycin inhibited the enzyme, while the blocker of Na+,K+-ATPase ouabain and the blocker of Ca2+,Mg2+-ATPase ruthenium red had no effect. The properties of the Cl−-stimulated Mg2+-ATPase of fish brain are discussed in comparison with those of the rat brain Cl−-ATPase. The conclusion is made that the bream brain enzyme differs markedly from Cl−-ATPase (the ATP-dependent Cl−-pump) of mammalian brain.