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The concentration of various anions in the sucrose impermeable space of ratliver mitochondria relative to the applied concentration can be used to calculate an equilibrium potential if one supposes that the anion distribution is passive. Values so calculated for anions of different net charge are about 30 mV, with the interior positive. The relative merits of the hypotheses that an internal positivity is generated by active cation movement or Mitchell 's chemiosmotic generation of an internal negativity coupled with hydroxyl-for-anion exchanges are examined.

Potentiometric techniques have been used to examine the interactions of certain redox dyes with mitochondria. Most of the dyes tested proved unsuitable in low concentrations for analysis by potentiometry, and, at high concentrations, they either had untoward effects on the mitochondria or served themselves as electron transfer reagents. However, tetramethyl p -phenylenediamine proved an exception in that it gave a rapid response at the platinum electrode at 10 μ m concentration and equilibrated rapidly with a component in the electron transfer system. Since this reagent was used in catalytic concentrations, it did not serve as a donor or sink of electrons, but rather as an indicator of the redox potential of a component of the mitochondria during respiration. Analysis of potential changes on adding ADP, inhibitors, or uncouplers, and comparison with spectroscopic data, enabled a correlation of the potential of TMPD with the response of cytochrome c . Polarographic analysis facilitated the examination of redox reagents which did not react rapidly in potentiometric analysis. The specificity of the reagent toward particular components of the mitochondrial chain depended on the reagent used. Vitamin K 5 proved to have a preferential specificity toward cytochrome b as shown by its polarographic response to the addition of reagents that alter the electron transfer characteristics of the mitochondria.

Washed, starved preparations of Escherichia coli B metabolize glucose with concomitant uptake of K + and O 2 and extrusion of H + . Abrupt changes in the rates of these processes are correlated with apparent sequential shifting in time of the control sites of aerobic glycolysis from the glyceraldehyde-3-phosphate dehydrogenase-3-phosphoglyceric phosphokinase couple to phosphofructokinase to hexikinase. It is suggested that the principal inhibitors of phosphofructokinase activity are citrate and ATP. The rate of K + uptake is neither a function of the rate of glucose dissimilation nor of the concentration of ATP in these short-term experiments, and the energy available for the active transport of ions appears to vary as the intracellular chemical environment changes.

THE possibility of reversing energy-dependent ion transport to synthesize ATP1,2 has apparently been realized in the case of chloroplasts subjected to acid-base transitions3. In no other case has net ATP synthesis coupled to ion transport been reported although incorporation of phosphorus-32 into ATP has been shown to be enhanced in erythrocytes by adjusting the sodium and potassium gradients so as to “reverse the Na+-pump”4 and during step-changes in pH applied to mitochondria5. The high apparent efficiency of the accumulation of potassium supported by ATP in mitochondria treated with the antibiotic valinomycin6 suggested that, provided the kinetic barrier was not inordinately large, ATP synthesis should be demonstrable in the absence of respiration if the direction of net potassium movement were reversed (that is, during potassium efflux).

In previous work a heat-stable, acetone-soluble fraction from rat liver microsomes was found to stimulate the respiration of mitochondria. By means of an assay system based on the activation of latent mitochondrial ATPase, the active component of the microsomal extract has been identified as free fatty acids. Among the saturated fatty acids, myristic exhibited the greatest activity which diminished progressively as the chain was lengthened or shortened. Introduction of a cis unsaturated bond, but not of a trans unsaturation, enhanced activity. During the interaction, the higher fatty acids or DCA are tightly bound by the mitochondria in contrast to DNP which activates latent ATPase without becoming appreciably bound.