1. A coupling factor has been purified from an acetone powder extract of Rhodospirilluin rubruin chromatophores. This restores to decoupled chromatophores (extracted with respect to coupling factor) both photophosphorylation and Mg2+-dependent ATPase activities. The coupling factor had been partially purified by gel filtration through a Sepharose 6B column, and further purified by ultra-filtration and sucrose-density-gradient centrifugation. At each stage of purification, the coupling factor activity was coincident with a Ca2+-dependent ATPase activity, increasing in total activity during the purification. Mg2+, Mn2+, Co2+ or Zn2+ could not substitute for Ca2+ in activation of this soluble ATPase, but these four metals strongly inhibited the activity of the Ca2+-dependent ATPase.2 . By incubation with decoupled chromatophores, the soluble Ca2+-dependent ATPase was rebound to the membrane in the presence of either Mg2+ or CaZ+. This binding resulted in a partial masking of the Ca2+-dependent ATPase activity of the soluble enzyme.
4.The membrane-bound Mg2+-or Ca2+-dependent ATPase activities were inhibited by oligomycin. Azide and parahydroxymercuribenzoate inhibited both the soluble and the membrane-bound ATPase activities. Treatment with phospholipase A inhibited both the membranebound ATPases, but had no effect on the soluble Ca2+-dependent ATPase.
.The sedimentation constant a t 1 mg protein/ml of the extensively purified coupling factor is s20,w = 13.1 x l O -l 3 s, and the approximate molecular weight 350000.ATPase activities are widely distributed among many types of biological membranes. Those associated with energy conversion systems coupled to electron transport respond specifically to uncouplers and energy-transfer inhibitors. A purified coupling factor from mitochondria has been demonstrated to display a Mg2+-dependent ATPase [l, 21. This provided strong evidence for a close relationship between ATPase activity and the electron-transport-coupled
The extent of hydrogen ion gradient across membranes from photosynthetic bacteria has been determined measuring the energy-dependent quenching of fluorescence of 9-aminoacridine in different experimental conditions. Examinations of the absorption or emission spectra of 9-aminoacridine in aqueous solution reveal an ideal behaviour of this amine up to millimolar concentrations.The decrease in fluorescence observed in the presence of energized membranes corresponds to a general quenching in all regions of the absorption or emission spectra and is related to the amount of dye and of vesicles present in the assay according to the model of distribution of an ideal amine. 4 The onset of a pH difference across the membrane is stimulated by the presence in the assay of lipophilic anions or of K+ and valinomycin; in these conditions a parallel decrease of membrane potential, as measured by carotenoid band shift, has been observed. The total protonmotive force evaluated by a combination of these two techniques however remains relatively constant during time and in almost all conditions tested.The quantitative test of the chemiosmotic coupling hypothesis requires a precise determination of the pH difference across the external and internal compartment of phosphorylating membrane vesicles.Methods developed and applied in intact mitochondria [l] and bacterial chromatophores [2] involve the use of pH-or cation-sensitive electrodes and often present technical difficulties since they require the evaluation of parameters, such as the internal buffering capacity of the particles or the internal concentration of free K+, which are affected by a great deal of uncertainty. Moreover they are not easily applied to small vesicular preparations such as submitochondrial or subchloroplast particles and chromatophores from phosphosynthetic bacteria.Methods, based on the distribution of weak acids [3] or amines [4--61 following the development of transmembrane pH difference offer significative advantages. Evaluation of dpH, obtained measuring the distribution of radioactive amines in chloroplasts [4], which are characterized by an active uptake of protons, are based on the assumption that only the uncharged species of the amine is freely permeable across the membrane [7,8]. Under this assumption the following relation holds :This general behaviour of amines has been suggested to be the mechanism which explains the energydependent quenching of fluorescence of certain aromatic amines such as atebrine and 9-aminoacridine [9,10], which have been used recently in chloroplasts for a quantitative evaluation of dpH [5].The validity of this model has been recently verified by Deamer et al. [ l l ] using phospholipid liposomes in which controlled pH gradients were established by different experimental approaches; their results show that 9-aminoacridine behaves ideally in this artificial membrane system as the model proposes.This method appears particularly useful for evaluating hydrogen ion gradients in bacterial membrane fragments since it allows a ...
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