Affinity chromatography of H+-translocating adenosine triphosphatase isolated by chloroform extraction of Rhodospirillum rubrum chromatophores. Modification of binding affinity by divalent cations and activating anions

1. ATPase isolated from Rhodospirillum rubrum by chloroform extraction and purified by gel filtration or affinity chromatography shows three bands (α, β and γ) upon electrophoresis in sodium dodecyl sulphate.2. Ca²⁺-ATPase activity of the preparation is inhibited by aurovertin and efrapeptin but not by oligomycin. Activity may be inhibited by treatment with 4-chloro-7-nitrobenzofurazan and subsequently restored by dithiothreitol.3. The enzyme fails to reconstitute photophosphorylation in chromatophores depleted of ATPase by sonic irradiation.4. Most of the active protein from the crude chloroform extract binds to an affinity chromatography column bearing an immobilised ADP analogue but not to a column bearing immobilised pyrophosphate.5. In the absence of divalent cations, a component with a very high specific activity for Ca²⁺-ATPase is eluted from the column by 1.6 mM ATP. This protein migrates as a single band on 5% polyacrylamide gel electrophoresis and only possesses three subunits. At 12 mM ATP an inactive protein is eluted which does not run on acid or alkali polyacrylamide gels and shows a complex subunit structure.6. ATPase preparations prepared by acetone extraction or by sonic irradiation of chromatophores may also be purified 10-fold by affinity chromatography.7. The inclusion of 5 mM MgCl₂ or CaCl₂ during affinity chromatography of chloroform ATPase increases the capacity of the column for the enzyme and demands a higher eluting concentration of ATP.8. When the enzyme is more than 90% inhibited by efrapeptin or 4-chloro-7-nitrobenzofurazan, the binding characteristics of the enzyme are not affected.9. 10 mM Na₂SO₃, which greatly stimulates the Ca²⁺- and Mg²⁺-dependent ATPase activity of the enzyme and increases K_i (ADP) for Ca²⁺-ATPase from 50 to 850 μM, prevents binding to the affinity column. Binding may be restored by the addition of divalent cations.10. Na₂SO₃ increases the rate of ATP hydrolysis, ATP-driven H⁺ translocation and ATP-driven transhydrogenase in chromatophores.11. It is proposed that anions such as sulphite convert the chromatophore ATPase into a form which is a more efficient energy transducer.