Selective Extraction and Reconstitution of F₁ Subunits from Rhodospirillum Rubrum Chromatophores

his chapter describes the selective extraction and reconstitution of F₁ subunits from Rhodospirillurn rubrum chromatophores. The F₁-ATPase isolated from membranes of mitochondria, bacteria, and chloroplasts is very similar, containing five nonidentical polypeptide subunits: α, β, γ, δ, and ε. Using R. rubrum chromatophores, two F₁ subunits—β and γ—are extracted in two consecutive steps, leaving all other F1 subunits attached to the chromatophore membrane. The procedures developed for the extractions, for purification of the isolated subunits, and for their reconstitution into the depleted chromatophores are described. The isolated β and γ subunits have no activity by themselves, although their extraction leads to complete loss of photophosphorylation and ATPase activities of the depleted chromatophores. The subunits are therefore identified during the extraction and purification procedures by their reconstitutive activity. This is their capacity to rebind to depleted chromatophores and restore their lost activities. The experimental system for measuring the reconstitutive activity involves two steps: (1) reconstitution of the isolated subunits into the depleted chromatophores, and (2) assay of the reconstituted chromatophores for restored activities.