బయోకెమిస్ట్రీ & మాలిక్యులర్ బయాలజీ జర్నల్ అందరికి ప్రవేశం

నైరూప్య

The Detection of a GABAAR β3 Subunit in an Affinity-Purified Preparation of the GABAAR-Associated Cl-/HCO3 --ATPase Isolated from Rat Brain

Sergey A. Menzikov

Background: The Cl^-/HCO₃^--ATPase from animal brain is a new primary active Cl^-- transport system that is coupled to the GABA_A receptor (GABA_AR). The structural coupling of the enzyme with GABA_AR, however, has not been confirmed by the presence of GABA_AR subunits within the purified protein complex.

Objective/Design: Identification of the enzyme using methodological approaches that would allow the demonstration of GABA_AR peptide subunits within a purified enzyme preparation.

Main outcome measures: A plasma membrane-enriched preparation (PMP) and a purified enzyme preparation (PEP) from rat brain were loaded onto high resolution clear native-PAGE (hrCNE-PAGE) gels and analyzed on a denaturing gel system. The Cl^-/HCO₃^--ATPase activity was detected in gel strips in the presence of Mg²⁺-ATP and 5 mM Cl^-/25 mM HCO₃^-, and was inhibited by ethanol (500 mM). The enzyme in the gel strips was detected by western blotting with antibodies against GABA_AR 1-3 subunit.

Results: The PEP gave one band with a molecular weight of 300 kDa. HrCNEPAGE/ SDS and western blotting results confirmed the presence of the GABA_AR β 1-3 subunits in the PMP. The PEP showed two subunits with molecular masses of 52 kDa and 57 kDa that were immuno-reactive only with GABA_AR ₃ antibodies. These peptide sub-units were directly phosphorylated by γ^-32 P-ATP and dephosphorylated in the presence of Cl^-/HCO₃^-, hydroxylamine and ethanol.

Conclusion: This proteomic approach and western blot analysis allowed the detection of the Cl^-/HCO₃^--АTPase along with GABA_AR ₃ subunits isolated from rat brain.