Effect of carbonic anhydrase inhibitors on enzymatic activity of isolated thylakoid CF1 ATPase

Abstract

We studied the effect of carbonic anhydrase inhibitors — acetazolamide (AA) and ethoxyzolamide (EA) — on the enzymatic activity of the isolated coupling factor CF₁ — a catalytic part of ATPsynthase complex of chloroplasts. The enzyme was isolated from spinach chloroplasts after their extraction with 1 mM EDTA. Carbonic anhydrase activity CF₁, which was determined in a solution by the acceleration of the formation of CO₂ in the bicarbonate dehydration reaction, was 73 μmol CO₂ · (min · mg protein)^-1 and almost 3 times more than the ATPase activity of the enzyme. Acetazolamide and ethoxyzolamide inhibit both the ATPase and carbonic anhydrase activities of CF₁· I₅₀ for Ca²⁺ -ATPase reaction catalyzed by isolated CF₁ in solution was 2 μM for AA and EA. ATPase activity increased somewhat with the concentration of EA. 50% inhibition of the carbonic anhydrase activity was achieved in the presence of 2 μM AA and 12 μM EA, respectively. Thus, water-soluble AA and liposoluble EA inhibit both the ATPase and carbonic anhydrase enzyme activities at similar and relatively low concentrations. The functional role of the discovered carbonic anhydrase activity may consist in facilitating the transfer of protons uptaken or released in the reactions of ATP synthesis or hydrolysis, respectively.