1. Pulses of acidity of the outer aqueous phase of rat liver mitochondrial suspensions induced by pulses of respiration are due to the translocation of H+ (or OH−) ions across the osmotic barrier (M phase) of the cristae membrane and cannot be attributed to the formation (with acid production) of a chemical intermediate that subsequently decomposes. 2. The effective quantity of protons translocated per bivalent reducing equivalent passing through the succinate-oxidizing and β-hydroxybutyrate-oxidizing spans of the respiratory chain are very close to 4 and 6 respectively. These quotients are constant between pH5·5 and 8·5 and are independent of changes in the ionic composition of the mitochondrial suspension medium provided that the conditions permit the accurate experimental measurement of the proton translocation. 3. Apparent changes in the →H+/O quotients may be induced by conditions preventing the occurrence of the usual backlash; these apparent changes of →H+/O are attributable to a very fast electrically driven component of the decay of the acid pulses that is not included in the experimental extrapolations. 4. Apparent changes in the →H+/O quotients may also be induced by the presence of anions, such as succinate, malonate and phosphate, or by cations such as Na+. These apparent changes of →H+/O are due to an increase in the rate of the pH-driven decay of the acid pulses. 5. The uncoupling agents, 2,4-dinitrophenol, carbonyl cyanide p-trifluoromethoxyphenylhydrazone and gramicidin increase the effective proton conductance of the M phase and thus increase the rate of decay of the respiration-driven acid pulses, but do not change the initial →H+/O quotients. The increase in effective proton conductance of the M phase caused by these uncouplers accounts quantitatively for their uncoupling action; and the fact that the initial →H+/O quotients are unchanged shows that uncoupler-sensitive chemical intermediates do not exist between the respiratory-chain system and the effective proton-translocating mechanism. 6. Stoicheiometric acid–base changes associated with the activity of the regions of the respiratory chain on the oxygen side of the rotenone- and antimycin A-sensitive sites gives experimental support for a suggested configuration of loop 3.

1. Pulsed acid-base titrations of suspensions of rat-liver mitochondria under anaerobic equilibrium conditions show fast and slow titration processes. 2. The fast process is the titration of the outer aqueous phase of the mitochondria, which is continuous with the suspension medium, and the slow process can be identified with the titration of the inner aqueous phase of the mitochondria, which is separated from the outer aqueous phase by the non-aqueous osmotic barrier or M phase of the cristae membrane system. 3. The buffering power of the outer and inner phases have been separately measured over a range of pH values. 4. The rate of titration of the inner aqueous phase under a known protonmotive force across the M phase has been characterized by an effective proton conductance coefficient, which, near pH7 and at 25 degrees , is only 0.45mumho/cm.(2) of the M-phase membrane. 5. The low effective proton conductance of the M phase will account quantitatively for the observed respiratory control in state 4, assuming that oxidoreduction and phosphorylation are coupled by a circulating proton current as required by the chemi-osmotic hypothesis. 6. The addition of 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) at normal uncoupling concentrations causes a large increase in the effective proton conductance of the M phase of the cristae membrane. 7. The increase of the effective proton conductance of the M phase by 2,4-dinitrophenol (or carbonyl cyanide p-trifluoromethoxyphenylhydrazone) will account quantitatively for the short-circuiting effect of the uncoupling agent on the proton current and for the observed rise of the rate of respiration to that characteristic of state 3 or higher.

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Studies of net translocation of various anions and cations and their corresponding acids and bases, using extensions of established techniques, imply that the M phase of the cristae membrane of rat liver mitochondria is very impermeable (i. e. not permitting net permeation) to the following ions: CH,COO-, H,PO;, HPOi-, SO:-, Fe(CN)i-, Fe(CN):-, choline+, K+, Na+ and NH:. However, the M phase appears to be permeated rapidly by SCN-and slowly by C1-.It has been confirmed that CH3COO-and "Ha probably permeate as CH3COOH and NH, respectively, and that phosphate equilibrates across the M phase by an electrically neutral mechanism equivalent to H,PO, translocation. The possibility that phosphate is obligatorily translocated as KKPO, has been eliminated.Sulphate equilibrates by an electrically neutral mechanism equivalent to H,SO, translocation. The alkali metals Na+ and K+ (and possibly N H f ) equilibrate across the M phase via specific electrically neutral H+/Na+ and H+/K+ antiport, the former being the more active a t neutral pH. Choline does not participate significantly in proton-coupled antiport.When resting rat liver mitochondria are suspended in isotonic solutions of the ammonium salts of various acids, the net rate of entry of the salt, and the rate of swelling observed semi-quantitatively by the change of light-sca,ttering, have been found by Chappell and co-workers [ -141 to depend on the acid or anionic component of the salt. It has been assumed [4] that neither NH: ion nor the charged anion An-can permeate the membrane as such, but that NH, can dissolve in and pass through the hydrophobic osmotic barrier (M phase) of the cristae membrane, and that net entry of salt is therefore conditional on a process equivalent to the entry of the acid AH,, as illustrated by the following translocation equations in which curved arrows denote chemical reaction and straight arrows denote translocation : H20where the anion AH&l, is written as 4-for simplicity, and where the circle between the two straight arrows denotes direct coupling between the corresponding translocation reactions. However, taking into account the osmotic equilibration of H,O across the membrane, the net translocation reaction of equations ( 2 ) and ( Chappell [a] has drawn the conclusion that the cristae membrane is anion-and cation-impermeable in the usual (uniport) sense, but that certain anions, such as arsenate, phosphate and acetate equilibrate across the cristae membrane of rat liver mitochondria by non-electrogenic reactions corresponding to acid uniport as described by equations ( 2 ) or (3) (see [6] for nomenclature), whereas other anions, such as chloride or sulphate do not equilibrate by such reactions. Chappell and Haarhoff [2] state that, unlike the ammonium salts, the sodium and potassium salts of phosphoric and acetic acid do not undergo net translocation through the cristae membrane. According to the chemiosmotic hypothesis [5,7,8], Na+ and K+

SUMMARY: The cell wall of exponential phase Staphylococcus aureus (strain Duncan) loses its tensile strength in c. 2 hr. when the organisms are incubated a t 25" in 1.2 M-sucrose a t pH 5.8 and ionic strength 0.3. The 'protoplasts' thus released from the mechanical protection of the cell wall are stable in 1.2 M-sucrose but lyse in media of lower osmotic pressure. The mean internal osmotic pressure of the 'protoplasts' is c. 20 atmospheres; they are permeable to glycerol but not to sucrose or NaCl. The rate of 'protoplast' release varies with the rate of growth of the organisms at harvesting. After osmotic explosion of 'protoplasts ' released from slowly growing organisms the plasma membranes may be recovered as spherical shells which disintegrate and condense into small particles on washing, and 75 yo of the weight of the cell walls may be recovered as hemispherical shells.The aim of the work described in this paper was to develop a method for releasing the protoplasts of Staphylococcus aureus (strain Duncan) from the protection of the cell wall so as to facilitate the study of the structure and function of the plasma membrane (Mitchell & Moyle, 1 9 5 6~) . Since staphylococci had been reported to be somewhat sensitive to lysozyme under certain conditions (Webb, 1948;Kern, Kingkade, Kern & Behrens, 1951) we decided to investigate the possibility of releasing stable ' protoplasts ' of staphylococci by modifications of the technique of Weibull (1953) similar to those which we developed for the release of the protoplasts of Il.l;icrococcus Zysodei&icus and Sarcina Zutea (Mitchell & Moyle, 19563). METHODSGrowth and preparation of organisms. Cultures were grown at 25" in a medium containing 3 yo (w/v) tryptic digest of casein, 1 yo glucose and 0.1 yo Marmite, either in Roux bottles or using the rotated flask technique (Mitchell, 1949). The organisms were harvested by centrifugation, washed once with distilled water and suspended at a concentration corresponding to c. 100mg. dry weightlml. in distilled water.Measurement of turbidity and dry weight. The extinction of 1 cm. depth of bacterial suspension was measured at a wavelength of 700mp., using the Beckman model DU spectrophotometer. The suspensions were diluted to give readings of log,, ( l o / I ) between 0.1 and 0.2 so that, the extinction being proportional to dry weight in this range, the dry weight could be estimated by multiplying the extinction by a factor. The factors for the different organisms were determined by calibrating the extinction against the dry-weight concentration estimated by drying c. 100 mg. samples to constant weight at 105O.

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