Carbodiimide-Binding Protein of H⁺-Translocating ATPase and Inhibition of H⁺ Conduction by Dicyclohexylcarbodiimide

Abstract

H⁺-Translocating ATPase, which catalyzes ATP synthesis in biomembranes, is composed of a head piece (F₁) and a membrane moiety (F₀). Using highly-purified Fo from a thermophilic bacterium PS3 (TF₀), the following results were obtained.
1. Inhibition by N, N'-dicyclohexylcarbodiimide (DCCD) of H⁺ conduction through TF₀ followed pseudo-first-order kinetics. The second-order rate constant for inhibitor-enzyme interaction was 5×10³M^-1•min^-1
2. H⁺ conductivity blocked by DCCD was proportional to the amount of DCCD incorporated in the band 8 protein of TF₀. When only one-third of the band 8 protein was labeled with DCCD, TF₀ hardly transported any H⁺.
3. By extracting TFo with chloroform-methanol, the band 8 protein was obtained as a proteolipid. Polyacrylamide gel electrophoresis with dodecyl sulfate and urea showed that the molecular weight was about 6, 000.
4. The amino acid composition of band 8 protein indicated that this protein contained an extremely high percentage of hydrophobic amino acids (0.29 in polarity) and was devoid of histidine, tryptophan, cysteine, and lysine. Its minimum molecular weight was 6, 500.
5. The role of band 8 protein (DCCD-binding protein) in H⁺ conduction through TF₀ is discussed on the basis of these results.