Effect of phenol on the GABAR-coupled Cl/HCO-ATPase from fish brain: An in vitro approach on the enzyme function.

Phenol (CHOH) has a toxic effect on the central nervous system of animals and humans. The Cl/HCO-ATPase from the plasma membranes of animal brains is the primary active P-type Cl-transporting system that is coupled to GABA receptor (GABAR). In this paper, we used an in vitro approach to assess the effects of phenol (1-500μM) on the functional parameters of the Cl/HCO-ATPase isolated from the fish brain. The enzyme is insensitive to phenol in the presence of Cl or HCO in the incubation medium. By contrast, in the presence of Cl/HCO, phenol inhibits (I=27μM) both the enzyme activity and its participation in ATP-dependent Cl transport through the membranes of artificial liposomes. Enriched plasma membranes and purified enzyme preparations were separated using hrCNE-PAGE. The ATPase activity in native gels was detected in the presence of phenol (100μM). Detection of ATPase activity in a purified preparation, showed a native protein of 300kDa, in agreement with western blot analysis with antibodies against GABAR β3 subunits. SDS-PAGE showed that one subunit with a molecular weight of 56kDa was directly phosphorylated by γ-P-ATP and dephosphorylated in the presence of phenol. The in vitro approach described in this work allowed the first demonstration that GABAR-coupled Cl/HCO-ATPase can be a protein marker for assessment of the toxicity of phenolics on the central nervous system.