The phosphorus/oxygen ratio of mitochondrial oxidative phosphorylation.

The transport of ATP out of mitochondria and uptake of ADP and Pi into the matrix are coupled to the uptake of one proton (Klingenberg, M., and Rottenberg, H. (1977) Eur. J. Biochem. 73, 125--130). According to the chemiosmotic hypothesis of oxidative phosphorylation this coupling of nucleotide and Pi transport to proton transport implies that the P/O ratio for the synthesis and transport of ATP to the external medium is less than the P/O ratio for the synthesis of ATP inside mitochondria. A survey of previous determinations of the P/O ratio of intact mitochondria showed little convincing evidence in support of the currently accepted values of 3 with NADH-linked substrates and 2 with succinate. We have measured P/O ratios in rat liver mitochondria by the ADP pulse method and by 32 Pi esterification, measuring oxygen uptake with an oxygen electrode, and find values close to 2 with beta-hydroxybutyrate as substrate and 1.3 with succinate as substrate in the presence of rotenone to inhibit NADH oxidation. These values were largely independent of pH, temperature, Mg2+ ion concentration, Pi concentration, ADP pulse size, or amount of mitochondria used. We suggest that these are the true values of the P/O ratio for ATP synthesis and transport by mitochondria, and that previously reported higher values resulted from errors in the determination of oxygen uptake and the use of substrates which lead to ATP synthesis by succinate thiokinase.     

The microbiol metabolism of C1 compounds. Oxidative phosphorylation in membrane preparations of Pseudomonas AM1

A method is described for preparation of membrane vesicles (diameter 80nm) capable of respiration-linked ATP synthesis. Vesicles prepared from succinate-grown bacteria oxidized NADH, succinate and ascorbate plus NNN'N'-tetramethylphenylenediamine; vesicles prepared from methanol-grown bacteria also oxidized methanol and formaldehyde, but they were otherwise identical. The uncoupling agent carbonyl cyanide chlorophenylhydrazone and the adenosine triphosphatase inhibitor dicyclohexylcarbodi-imide both inhibited ATP synthesis, whereas they had no effect on the rate of respiration. Rotenone inhibited ATP synthesis and respiration with NADH as substrate; antimycin A inhibited with succinate as substrate, and cyanide inhibited with all substrates. P/O ratios were usually 0.7-1.3 with NADH, 0.6-1.0 with succinate and 0.2-0.6 with reduced NNN'N'-tetramethylphenylenediamine or methanol as respiratory substrate. When 2,6-dichlorophenol-indophenol was used as an alternative electron acceptor to O(2) (NADH as donor) the P/2e ratio was 1.65. Although these P/O ratios are minimum values, because they do not take into account unknown amounts of uncoupled O(2) consumption, they are consistent with previous proposals [O'Keeffe & Anthony (1978) Biochem, J.170, 561-567] based on measurements of proton translocation in whole cells. The results also confirm that methanol dehydrogenase and cytochromes c and a/a(3) are arranged so that the first step in methanol oxidation is coupled to synthesis of ATP.     

The upper and lower limits of the mechanistic stoichiometry of mitochondrial oxidative phosphorylation. Stoichiometry of oxidative phosphorylation

Determination of the intrinsic or mechanistic P/O ratio of oxidative phosphorylation is difficult because of the unknown magnitude of leak fluxes. Applying a new approach developed to overcome this problem (see our preceding paper in this journal), the relationships between the rate of O2 uptake [( Jo)3], the net rate of phosphorylation (Jp), the P/O ratio, and the respiratory control ratio (RCR) have been determined in rat liver mitochondria when the rate of phosphorylation was systematically varied by three specific means. (a) When phosphorylation is titrated with carboxyatractyloside, linear relationships are observed between Jp and (Jo)3. These data indicate that the upper limit of the mechanistic P/O ratio is 1.80 for succinate and 2.90 for 3-hydroxybutyrate oxidation. (b) Titration with malonate or antimycin yields linear relationships between Jp and (Jo)3. These data give the lower limit of the mechanistic P/O ratio of 1.63 for succinate and 2.66 for 3-hydroxybutyrate oxidation. (c) Titration with a protonophore yields linear relationships between Jp, (Jo)3, and (Jo)4 and between P/O and 1/RCR. Extrapolation of the P/O ratio to 1/RCR = 0 yields P/O ratios of 1.75 for succinate and 2.73 for 3-hydroxybutyrate oxidation which must be equal to or greater than the mechanistic stoichiometry. When published values for the H+/O and H+/ATP ejection ratios are taken into consideration, these measurements suggest that the mechanistic P/O ratio is 1.75 for succinate oxidation and 2.75 for NADH oxidation.     

The ATP-to-oxygen stoichiometries of oxidative phosphorylation by rat liver mitochondria. An analysis of ADP-induced oxygen jumps by linear nonequilibrium thermodynamics

Uncertainty exists as to the proton stoichiometries of mitochondrial oxidative phosphorylation and consequently as to the ATP stoichiometries. In rat liver mitochondria, ADP/O ratios were determined from the total and extra oxygen consumed during ADP-stimulated respiration under conditions of quantitative conversion of ADP to ATP. For succinate, glutamate plus malate, 3-hydroxybutyrate, and 2-oxoglutarate, respectively, ADP/total O was 1.71, 2.71, 2.61, and 3.45. ADP/extra O was 2.03, 3.04, 3.23, and 4.15. The results were interpreted in terms of linear nonequilibrium thermodynamics. It was shown that ADP/extra O = Z/q where Z is the phenomenological stoichiometry and q is the degree of coupling. q was determined from the dependence of respiratory rate on delta Gp, the phosphorylation potential, and was about 0.98 for all substrates. The results were consistent with ideal ATP/O stoichiometries of 2 for succinate, 3 for glutamate plus malate, 3 or 3 1/4 for 3-hydroxybutyrate, and 4 for 2-oxoglutarate. Taking into account the oxidation-reduction free-energy changes measured across Sites 1 + 2 at static head (J.J. Lemasters, R. Grunwald, and R.K. Emaus J. Biol. Chem. 259, 3058-3063), an ideal ATP/O stoichiometry of 3 1/4 for 3-hydroxybutyrate is proposed. The lower ATP/O for glutamate plus malate is then accounted for by proton translocation linked to glutamate/aspartate exchange. The data suggest a new 13-proton scheme of chemiosmotic coupling in which proton stoichiometries are 3 for the F1Fo-ATPase, 1 for the exchange of ATP for ADP and Pi, and 5, 4, and 4 for Sites 1, 2, and 3.     

Effect of corticosterone treatment on energy metabolism in rat liver mitochondria.

1. Effect of in vivo treatment (40 mg/kg body wt) with corticosterone on energy metabolism in rat liver mitochondria was examined under acute and chronic conditions in 20-, 35- and 60-day-old rats. 2. Acute treatment did not affect body or liver weight. However, chronic treatment caused increased liver weight in the former two age groups; in the 60-day-old animals the liver weight decreased. 3. Acute treatment resulted in a generalized decrease in state 3 respiration rates and state 4 respiration rates without having any significant effect on ADP/O ratios with glutamate, succinate and ascorbate + TMPD as substrates. However, rates of ATP synthesis decreased significantly. The effect was age-dependent, older animals showed increased resistance. 4. Chronic treatment resulted in uncoupling of oxidative phosphorylation without having significant effects on respiration rates. Once again, the effects were age-dependent. Consequently, the ATP synthesis rates were significantly lowered. However, it was apparent that the underlying mechanisms were entirely different. 5. With succinate as the substrate the state 3 respiration rates increased with age to reach adult values by day 60. The coupling efficiency was also exhibited via maturational changes.     

Phosphorylation coupled to O2 and nitrate reduction inPseudomonas saccharophila grown anaerobically with succinate and nitrate

Cell-free membrane preparations fromPseudomanas saccharophila grown anterobically with succinate and nitrate catalyzed NADH oxidation by O₂ and nitrate, yielding P/O and P/NO₃ ⁻-reduced ratios of 0.76 and 0.51, respectively. Succinate oxidation yielded a P/O ratio of 0.44 and a P/NO₃ ⁻-reduced ratio of 0.08. Ascorbate oxidation by O₂ or nitrate was not coupled with ATP generation. The NADH- or succinate-linked oxidative phosphorylation was uncoupled by classical uncoupling agents: moreover, the aerobic and the anaerobic oxidation of NADH and succinate, as well as the coupled ATP synthesis, was inhibited by low concentrations of respiratory chain inhibitors. In addition, oligomycin was a potent inhibitor of ATP generation in this system.     

Evidence for the rapid direct control both in vivo and in vitro of the efficiency of oxidative phosphorylation by 3,5,3''-tri-iodo-L-thyronine in rats.

1. Examination of the distribution of L-tri-iodothyronine among rat liver tissue fractions after its intravenous injection into thyroidectomized rats focused attention on mitochondria at very short times after administration. By 15 min this fraction contained 18.5% of the tissue pool; however, the content had decreased sharply by 60 min and even further over the next 3 h. By contrast, the content in all other fractions was constant or increased over 4 h. About 60% of tissue hormone was bound to soluble protein. 2. Mitochondria isolated from thyroidectomized rats showed P/O ratios that were about 50% of those found in normal controls, with both succinate and pyruvate plus malate as substrates. There was no evidence of uncoupling; the respiratory-control ratio was about 6. 3. Mitochondria isolated 15 min after injection of tri-iodothyronine into thyroidectomized rats showed P/O ratios and respiratory-control ratios that were indistinguishable from those obtained in mitochondria from euthyroid animals. The oxidation rate was, however, not restored. 4. Incubation of homogenates of livers taken from thyroidectomized animals injected with L-tri-iodothyronine before isolation of the mitochondria restored the P/O ratio to normal; by contrast, direct addition of hormone to isolated mitochondria had no effect. The role of extramitochondrial factors in rapid tri-iodothyronine action is discussed. 5. Possible mechanisms by which tri-iodothyronine might rapidly alter phosphorylation efficiency are considered: it is concluded that control of adenine nucleotide translocase is unlikely to be involved. 6. The amounts of adenine nucleotides in liver were measured both after thyroidectomy and 15 min after intravenous tri-iodo-thyronine administration to thyroidectomized animals. The concentrations found are consistent with a decreased phosphorylation efficiency in thyroidectomized animals. Tri-iodothyronine injection resulted in very significant changes in the amounts of ATP, ADP and AMP, and in the [ATP]/[ADP] ratio, consonant with those expected from an increased efficiency of ADP phosphorylation. This suggests that the changes seen in isolated mitochondria may indeed reflect a rapid response of liver in vivo to tri-iodo-thyronine.     

Mitochondrial coupling in vivo in mouse skeletal muscle

The coupling of mitochondrial ATP synthesis and oxygen consumption (ratio of ATP and oxygen fluxes, P/O) plays a central role in cellular bioenergetics. Reduced P/O values are associated with mitochondrial pathologies that can lead to reduced capacity for ATP synthesis and tissue degeneration. Previous work found a wide range of values for P/O in normal mitochondria. To measure mitochondrial coupling under physiological conditions, we have developed a procedure for determining the P/O of skeletal muscle in vivo. This technique measures ATPase and oxygen consumption rates during ischemia with ³¹P magnetic resonance and optical spectroscopy, respectively. This novel approach allows the independent quantitative measurement of ATPase and oxygen flux rates in intact tissue. The quantitative measurement of oxygen consumption is made possible by our ability to independently measure the saturations of hemoglobin (Hb) and myoglobin (Mb) from optical spectra. Our results indicate that the P/O in skeletal muscle of the mouse hindlimb measured in vivo is 2.16 ± 0.24. The theoretical P/O for resting muscle is 2.33. Systemic treatment with 2,4-dinitrophenol to partially uncouple mitochondria does not affect the ATPase rate in the mouse hindlimb but nearly doubles the rate of oxygen consumption, reducing in vivo P/O to 1.37 ± 0.22. These results indicate that only a small fraction of the oxygen consumption in resting mouse skeletal muscle is nonphosphorylating under physiological conditions, suggesting that mitochondria are more tightly coupled than previously thought. P/O; oxidative phosphorylation; proton leak; optical spectroscopy     

An assessment of the role of proton leaks in the mechanistic stoichiometry of oxidative phosphorylation.

Rat liver mitochondria were incubated in the presence of varying concentrations of ATP, followed by ADP to initiate phosphorylation. Analysis of phosphorylation to oxygen ratios (P/O) was carried out with varied initial phosphorylation potentials (or ATP/ADP ratios). Rates of phosphorylation and respiration and magnitude of membrane potential (delta psi) were measured. The results are discussed in the framework of P/total O and P/"extra" O ratios in determination of the mechanistic P/O ratio. It is concluded that the former underestimates, and the latter overestimates the mechanistic P/O ratio.     

Studies of Respiratory Components and Oxidative Phosphorylation in Mitochondria of mi-1 Neurospora crassa

Oxidative phosphorylation has been demonstrated with mitochondria of the mi-1 respiratory mutant of Neurospora crassa. The P/O ratios observed with these mitochondria were approximately 0.8 with citrate and 0.4 with either externally added reduced nicotinamide adenine dinucleotide (NADH), succinate, or ascorbate-tetramethyl-p-phenylenediamine (TPD). These P/O ratios suggest that there are only two sites of phosphorylation in mitochondria isolated from young (20 to 24 h) cultures of the mi-1 mutant. The energy-dependent reduction of NAD(+) with succinate and the phosphorylation associated with ascorbate-TPD oxidation indicate that the first and the third sites of energy coupling are present in this mutant. Difference spectra of mitochondria from young cultures of the mi-1 mutant revealed the presence of cytochrome c. Cytochromes b and a + a(3) were not detected. However, in the presence of antimycin A, a small peak in the Soret region at 430 nm was observed. A carbon monoxide difference spectrum revealed the presence of a component of the respiratory chain with a spectrum similar to that of cytochrome o. It is of interest that respiratory inhibitors such as antimycin A, 2-n-nonylhydroxyquinoline N-oxide, and cyanide abolished phosphorylation but only partially inhibited oxidation. It is postulated that the mi-1 respiratory system contains two pathways of electron transport-the first is associated with a phosphorylating pathway, whereas the second is a non-phosphorylating electron transport pathway.     

Energy conservation during nitrate respiration in Paracoccus denitrificans

P/2e ratios were calculated from anaerobic chemostat cultures of Paracoccus denitrificans with nitrogenous oxides as electron acceptor. P/2e ratios were calculated, using the Y _ATP ^max values determined for aerobic cultures. When succinate was the carbon and energy source the average P/2e values of the sulphate-and succinate-limited cultures with nitrate as electron acceptor were 0.5 and 0.7, respectively, and of the nitrite-limited culture 0.9. With gluconate as carbon and energy source the average P/2e values of the gluconate-limited with nitrate as electron acceptor and nitrate limited cultures were 0.9 and 1.1, respectively.H⁺/O ratios measured in cells obtained from sulphate-, succinate, nitrite-, gluconate-and nitratelimited cultures yielded respective average values of 3.4, 4.5, 3.5, 4.8 and 6.2 for endogenous substrates. From our data we conclude that sulphate-and nitritelimitation causes the loss of site I phosphorylation. Nitrite has no influence on the maximum growth yield on ATP. We propose that metabolism in heterotrophically grown cells of Paracoccus dentrificans is regulated on the level of phosphorylation in the site I region of the electron transport chain.     

Nutritional effects on mitochondrial bioenergetics. Alterations in oxidative phosphorylation by rat liver mitochondria.

Rats malnourished since birth and fed on a protein-free diet for 2 weeks showed a 23-27% decrease in the State-3 oxidation of glutamate, succinate and ascorbate + NNN' N'-tetramethyl-p-phenylenediamine by liver mitochondria compared with control fed animals. ATP synthesis and the respiratory control index were diminished at the three coupling sites, but significant alterations were not observed in ADP/O ratios. Vmax. for NADH oxidation in electron-transport particles was 40% lower. Mitochondrial cytochromes b and c1 remained unchanged, but cytochrome c was increased by 26%. Cytochromes a + a3 were diminished by 22%. Vmax. for mitochondrial ATPase was 23% lower. These results suggest that the lower content of cytochrome a + a3 at the rate-controlling step of oxidative phosphorylation in malnourished rats might be mainly responsible for the decrease in substrate oxidations as well as ATP synthesis at the three coupling sites. The decreased synthesis and hydrolysis of ATP suggests that other energy-dependent mitochondrial processes could be decreased during malnutrition.     

Mechanistic stoichiometry of mitochondrial oxidative phosphorylation

P/O ratios of rat liver mitochondria were measured with particular attention to systematic errors. Corrections for energy loss during oxidative phosphorylation were made by measurement of respiration as a function of mitochondrial membrane potential. The corrected values were close to 1, 0.5, and 1 at the three coupling sites, respectively. These values are consistent with recent measurements of mitochondrial proton transport.     

Human quadricepts muscle mitochondria: A functional characterization

Human quadriceps mitochondria were isolated from ca. 80 mg tissue in ca. 45% yield. The preparation is described with respect to content of mitochondrial markers and nine different respiratory activities. The specific state 3 activities were high in comparison with literature data, indicating high integrity and purity of the preparation. Examples of state 3 rates, in µmol O min^-1 g protein^-1 (25°C): pyruvate + malate, 400; succinate, 514; malate + glutamate, 444. The notion of high integrity was also supported by the reproducibility of the preparation and the magnitude of the respiratory control ratios and the P/O ratios. The mitochondria most likely had lost ca. 30% of their cytochrome c upon isolation, but it was substantiated that this loss had not influenced the state 3 rates. Functional assays of single reactions or groups of reactions could be based on respiration experiments. The respiratory chain activity, for instance, was measured as respiration of NADH in freeze-permeabilized mitochondria (1263 mol O min^-1 g protein^-1). Comparison of uncoupled rates of respiration and state 3 rates indicated that the ATP synthesis exerted major flux control over respiration of succinate + glutamate, malate + glutamate and pyruvate + malate. These reactions, showing very similar rates of ATP synthesis, could be used as a functional assay of ATP synthesis (1200 mol ATP min^-1 g protein^-1). Respiration of succinate, palmitoyl-carnitine + malate, or glutamate could not support the maximal rate of ATP synthesis and the upstream reactions probably exerted major flux control in these cases. The specific activities appeared very constant in this group of young men, only the respiratory activity with glutamate might show biological variation.     

Distinct physiological roles of animal succinate thiokinases. Association of guanine nucleotide-linked succinate thiokinase with ketone body utilization

Two distinct succinate thiokinases have recently been shown to exist in animal tissues, one specific for guanine nucleotide and the other for adenine nucleotide. Their physiological roles have here been investigated by comparing the levels of the two enzymes in liver and brain of normal and diabetic rats. A marked rise in the level of brain guanine nucleotide-linked succinate thiokinase in the diabetic condition is consistent with an enhanced utilization of ketone bodies and hence with the associated elevated demand for succinyl-CoA for the activation of acetoacetate. Taken together with the reported mitochondrial values of the ATP/ADP and GTP/GDP ratios, the results are interpreted to indicate that the adenine nucleotide-linked enzyme functions as a component of the citric acid cycle whereas the guanine nucleotide-linked enzyme functions in the opposite metabolic direction to produce succinyl-CoA from succinate.     

31P nuclear magnetic resonance and saturation transfer studies of the isolated perfused rat kidney

Previous studies using 31P nuclear magnetic resonance (NMR) saturation transfer techniques to quantitate the energy metabolism of the kidney have often resulted in estimates of adenosine triphosphate (ATP) turnover which are much lower than those predicted from the renal oxygen consumption and reasonable values of the P/O ratio. We measured the ATP turnover in isolated perfused kidneys of rats, using 31P NMR saturation transfer and a new procedure for quantitation of the intracellular Pi concentration. The estimated turnover rates of ATP were higher than previously reported. The P/O ratios calculated on the basis of these rates of ATP turnover and rates of renal oxygen consumption reported in the literature were within the range of theoretically possible values. Thus, 31P NMR saturation transfer can be used to quantitate the ATP turnover in the isolated perfused rat kidney.     

Theoretical calculations on the influence of the inorganic nitrogen source on parameters for aerobic growth of microorganisms

The amount of ATP required for the formation of microbial cells growing in a minimal medium with various nitrogen sources was calculated. In a glucose-mineral salts medium 28.8 g cells can be formed per mole ATP with ammonia and 23.1 for growth with nitrate. For growth with molecular nitrogen 11.1; 8.7; 7.1 and 6.0 g cells can be formed per mole ATP for ATP/N₂ ratios of 12, 18, 24 and 30 respectively. A method is given for the calculation of Y_sub, Y_O2 and Y_{c0 2} values for aerobic growth with glucose, succinate or methanol and various nitrogen sources. In this method use is made of the elementary composition of the cells and of mass balance equations. As an assimilation equation: C₆H₁₂O₇ + 1.4 HNO₃ + 6.85 “H₂” → C₆H_10.84N_1.4O_3.07 + 8.13 H₂O is given for growth ofParacoccus denitrificans with gluconate and nitrate. From this equation and the molar growth yield for gluconate the oxygen uptake, carbon dioxide evolution and the Y_O2 value can be calculated. A very good agreement between the calculated values and the experimental values was obtained. For the calculation of the ATP production it is essential to know the number of phosphorylation sites in the respiratory chain. Calculations are given for 2 (sites I + II) and 3 phosphorylation sites. The molar growth yields for growth with nitrate and nitrogen are much smaller than that for growth with ammonia. The Y_O2 values for growth with glucose and nitrate are higher (with 2 sites) or somewhat smaller (with 3 sites) than for growth with ammonia. The Y_O2 values for growth with nitrogen are always very low. The calculations show that especially Y_O2 is very dependent on the number of phosphorylation sites. For growth with methanol Y_{CO 2} is strongly dependent on the nature of the assimilation pathway for methanol and on the nitrogen source. The molar growth yields for growth with glucose, succinate or methanol and nitrogen are about the same as when nitrate is the nitrogen source for organisms with 3 phosphorylation sites. The theoretical efficiency of nitrogen fixation in grow-ing cells is much lower (dependent on the ATP/N₂ ratio) than that in nongrowing cells.     

Determination of the P/2e- stoichiometries at the individual coupling sites in mitochondrial oxidative phosphorylation. Evidence for maximum values of 1.0, 0.5, and 1.0 at sites 1, 2, and 3

P/2e- stoichiometries in six assay systems spanning different portions of the respiratory chain were estimated by direct determinations of Pi uptake in suspensions of bovine heart mitochondria containing a hexokinase trap. The electron donors were malate + pyruvate, succinate, and ascorbate + N,N,N',N'-tetramethyl-p-phenylenediamine, and the electron acceptors were ferricyanide (Site 1, Site 2, and Sites 1 + 2) and O2 (Sites 1 + 2 + 3, Sites 2 + 3, and Site 3). A major objective was to find conditions in which the six systems yield results in sufficiently good agreement to allow confidence as to their reliability. This objective was achieved, and maximum values of 1.1, 0.5, and 1.0 were observed in the Sites 1, 2, and 3 systems, respectively. This required that the energy-conserving reactions be relatively nonlimiting and that the P/2e- ratios be estimated from the slopes of plots of respiration rate versus phosphorylation rate obtained by inhibiting oxidative phosphorylation with respiratory chain inhibitors. The latter requirement allows avoidance of the effect of an apparent endogenous uncoupler and is based on the observation (Tsou, C. S., and Van Dam, K. (1969) Biochim. Biophys. Acta 172, 174-176) that uncoupling agents at low concentrations decrease the rate of phosphorylation nearly as much in absolute amount at low rates of respiration as at high rates. The maximum P/2e- stoichiometry at Site 1 is considered to be 1.0, and the value observed in the Site 1 system is suggested to be higher as a result of H+ ejection at the transhydrogenase level. Respiratory control due to carboxyatractyloside inhibition was examined and found to differ greatly among the systems. It is pointed out that this observation is not consistent with the lack of complete control being due primarily to ion cycling and that, in view of this, the relatively meager control at Site 3 is not consistent with O2 being reduced on the matrix side of the coupling membrane.     

Oxidative phosphorylation in Micrococcus denitrificans. I. Preparation and properties of phosphorylating membrane fragments

A procedure was described to prepare stable membrane fragments from aerobically grown cells of Micrococcus denitrificans. This preparation contained flavins, cytochromes b, c, a and o, and catalyzed the synthesis of ATP coupled to the oxidation of NADH and succinate. The P:O ratios were about 1.0 for NADH and 0.4 for succinate oxidation. The electron-transfer pathways responsible for these oxidations were similar to, though not identical with, those of mammalian mitochondria in their construction and sensitivity to inhibitors. Oxidative phosphorylation by the membrane fragments was uncoupled by the usual uncouplers and energy-transfer inhibitors, though 2,4-dinitrophenol was much less effective and higher concentrations of oligomycin and tributyltin chloride were required for complete inhibition as compared with the mitochondrial system. Oleate also caused uncoupling, which was relieved by serum albumin. Treatment with high concentrations of LiCl yielded an essentially uncoupled preparation, but this treatment as well as many other procedures failed to yield soluble coupling factors. Unlike the mitochondrial ATPase activity, ATP hydrolysis by the membrane fragments was inhibited to about 50% by uncouplers and energy-transfer inhibitors. It seems that the bacterial preparation possessed two types of ATPase, one of which was sensitive to these reagents as well as to LiCl treatment and probably to high concentrations of ADP. The advantage of this preparation for the study of the mechanism of oxidative phosphorylation is discussed.