Stoichiometry of H+ ejection during respiration-dependent accumulation of Ca2+ by rat liver mitochondria.

We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxtbutyrate were accumulated with each Ca2+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis.

We have investigated the energy-dependent uptake of Ca2+ by rat liver mitochondria with succinate as respiratory substrate and with rotenone added to block NAD-linked electron transport. In the presence of 3-hydroxybutyric or other permeant monocarboxylic acids Ca2+ was taken up to extents approaching those seen in the presence of phosphate. The quantitative relationship between cation and anion uptake was determined from the slope of a plot of 3-hydroxybutyrate uptake against Ca2+ uptake, a method which allowed determination of the stoichiometry without requiring ambiguous corrections for early nonenergized or nonstoichiometric binding events. This procedure showed that 2 molecules of 3-hydroxybutyrate were accumulated with each Ca '+ ion. Under these conditions close to 2 Ca2+ ions and 4 molecules of 3-hydroxybutyrate were accumulated per pair of electrons per energy-conserving site of the respiratory chain. Since 3-hydroxybutyrate must be protonated to pass the membrane as the undissociated free acid, it is concluded that 4 protons were ejected (and subsequently reabsorbed) per pair of electrons per energy-conserving site, in contrast to the value 2.0 postulated by the chemiosmotic hypothesis.
The 3-hydroxybutyrate/Ca'+ ratio was constant over a wide range of concentrations of Ca*+ and of 3-hydroxybutyrate and was independent of time of incubation and of extent of uptake. A number of other monocarboxylic acids showed the same stoichiometry demonstrating that the effect was not specific for 3-hydroxybutyrate.
Mitochondria isolated from malignant cells exhibited the same behavior under these conditions as those from normal liver.
The quantitative relationships between the transport of cations and the passage of electrons down the respiratory chain have been the object of much attention in the last decade, particularly with respect to Ca*+ and to H+ transport. The number of Ca2+ ions taken up per pair of electrons passing each energy-conserving site (the Ca*+/-ratio) normally appears to be close to 2 during respiration (l-5), although under special conditions "superstoichiometric" uptake is observed in which the Ca2+/-ratio may reach very high values (6)(7)(8)(9)(10)(11)(12)(13). The number of protons ejected per CaZ+ ion taken up (the H+/Ca2+ ratio) has often been reported to be 0.85 to 1.0 (1,7,8,14,15), although higher values approaching 2.0 have been observed (16,17). The H+/Ca'+ ratio depends in part on the nature of the anions in the system; for example, in the presence of acetate the H+/Ca2+ ratio falls to about 0.2 (15). These variations, together with the possible importance of the H+/-ratio in the mechanism of oxidative phosphorylation, have led us to reinvestigate the problem of the stoichiometry of H+ ejection during Ca*+ uptake.
Large amounts of Ca*+ may be accumulated by mitochon-*This work was supported by Grant BMS-72-02406 from the National Science Foundation, Grant GM-05919 from the National Institutes of Health, and Contract NOl-CP-45610 from the National Cancer Institute. dria together with certain anions, such as phosphate (18)(19)(20), acetate and anions of other monocarboxylic acids (15, 21-25), or bicarbonate (25, 26). Formally, each of these anions enters with a proton (26)(27)(28)(29)(30), in response to the alkalinity generated in the matrix by electron transport. Uptake of these anions, after correction for residual proton ejection, should be a measure of the number of protons ejected (and then largely carried back into the mitochondria) during uptake of Ca*+. With phosphate as the permeant anion Pi/Caz+ accumulation ratios of 0.6 have been observed (2,3), corresponding to accumulation within the mitochondrial matrix of insoluble calcium phosphates. With bicarbonate as anion source the CO,'-/Ca*+ accumulation ratio is 1.0 (26), corresponding to accumulation (and presumably precipitation) of 2 molecules of CaCO, per pair of electrons per site (26). Since phosphate and carbonate may occur in different ionic species depending upon the pH, calculation of the intrinsic H+/-ratio from data on these anions is hazardous since it is necessarily dependent upon assumptions about the intramitochondrial pH. On the other hand, simple monocarboxylic aliphatic acids are not subject to these complications; they have but a single ionizing group, their Ca2+ salts are extremely water-soluble and thus do not precipitate from solution in the matrix, and (in the presence of rotenone) their oxidative metabolism is negligible. Stoichiometry of 3-Hydroxybutyrate and Caz+ Accumulation 969 In this paper we describe experiments using 3-hydroxybutyric and a number of other lipophilic acids as a source of counteranions for the respiration-dependent accumulation of Ca*+. We have found that when nonspecific Cal+ binding and other effects are eliminated, the weak acid/Ca*+ accumulation ratio is close to 2.0, equivalent to an H+/Ca2+ ratio of 2.0 and an overall H+/-ratio of 4.0 for electron transport under conditions in which Ca2+ is being accumulated. was able to support swelling; however, this was abolished by subsequent addition of rotenone (Fig. 1D). Swelling was then restimulated by succinate, and once again prevented by antimycin A, showing the energy-dependent nature of swelling in the presence of Ca2+ and 3-hydroxybutyrate.
Uptake of Ca2+ and 3-Hydrorybutyrate-Both Ca2+ and 3-hydroxybutyrate were accumulated by the mitochondria under these conditions; this is shown in Fig. 2. Succinate was present as a source of reducing equivalents while oxidation and subsequent metabolism of the radiolabeled 3-hydroxybutyrate were prevented by rotenone. The reaction was initiated by addition of mitochondria from a stock suspension at 50 mg of protein/ml of sucrose and terminated by centrifugation to prevent ion movements. This took about 20 s; the time scale in the figures has been adjusted accordingly. Initiation of the reaction by Caz+ addition did not alter the effects observed. Uptake was measured by accumulation of radioactive label. It was corrected for the extra-matrix material in the pellet by determination of the sucrose-accessible space as described under "Experimental Procedure." In the absence of CaZ+ only a small amount ( <50 nmol/mg of protein) of X-hydroxybutyrate was taken up by the mitochondria. Similarly, Caa+ uptake in the absence of 3-hydroxybutyr-ate was relatively slight. However, when both 20 mM ,3-hydroxybutyrate and 1 mM CaCl, (400 nmol of Ca*+/mg of protein) were present, there was a very substantial uptake of both. 3-Hydroxybutyrate was accumulated to 450 nmol/mg of protein and Ca 2+ to 250 nmol/mg of protein with maximal uptake under these conditions after about 8 min of incubation. The subsequent slight decrease in accumulation was attributed to increasing membrane leakiness due to the prolonged incubation and increased volume of the mitochondrial matrix. Uptake of [2, was measured under these conditions and was found to be less than 27 nmol/mg of protein; the extra succinate uptake in the presence of Caa+ was 1esS than 10 nmol/mg. These amounts were considered to be negligible.
Stoichiometry of Uptake: the 3-HydroxybutyratelCaz+ Ratio -It is apparent from Fig. 2 that the time course of uptake was similar for Ca*+ and 3-hydroxybutyrate and that the ratio of acid to Ca*+ accumulated was about 2. In order to establish this relationship more exactly, the amount of 3-hydroxybutyrate taken up was plotted as a function of Ca2+ uptake for time intervals varying from 0.3 to 10 min; this is presented in Fig. 3. This method of calculating the stoichiometry was chosen as it allowed correction for early, nonenergized binding of ions while avoiding a number of complicating assumptions. It also allowed calculation of a best line through the points by orthogonal regression, with a slope equal to the ratio of nanomoles of 3-hydroxybutyrate accumulated to nanomoles of Ca*+ accumulated. Extra 3-hydroxybutyrate uptake was calculated by subtracting the small amount bound in the absence of Ca2+ from that bound in the presence of Ca2+ (Fig. 2, Line A minus Line D). Uptake of Ca?+ was not corrected in a similar way as: (a) Ca*+ uptake in the absence of 3-hydroxybutyrate was partially due to endogenous phosphate (such uptake would be expected to decrease in the presence of a large excess of the competing 3-hydroxybutyric acid) and (b) the addition of a permeant acid may allow membrane-bound CaZ+ to enter the mitochondrial matrix (15,20,(32)(33)(34) and invalidate the correction.
A slope of 2.04 with standard deviation 0.13 was calculated from the data in Fig. 3, indicating that for each Ca*+ ion taken 'r Tlmc (mln) FIG. 2. Time course of Cal+ and 3-hydroxybutyrate uptake. For details see "Experimental Procedure." 3-Hydroxybutyrate when present was 20 mM; Cal+ when present was 1 rn~ (400 nmol/mg of protein). Succinate, 2.5 mM, and rotenone, 2.5 PM, were also present. Temperature: 23'; pH 7.2. A, %Hydroxybutyrate uptake in the presence of Cal+; B, Ca*+ uptake in the presence of 3-hydroxybutyrate; C, CaZ+ uptake in the absence of added permeant acid; D, 3-Hydroxybutyrate uptake in the absence of added Caz+. up 2 molecules of 3-hydroxybutyrate were accumulated under these conditions. The intercept on the "Ca'+ bound" axis was 50 nmol/mg of protein; small variations in this value between mitochondrial preparations caused some of the scatter of points seen in Fig. 3.
Ca"+l-and 3-Hydroxybutyratel-Ratios-During uptake of Ca2+ in the presence of phosphate, Cal+/-ratios of 1.7 to 2.0 have been reported (l-5). In order to determine whether such a relationship also held during Ca2+ uptake supported by 3hydroxybutyrate we measured oxygen uptake in parallel experiments to those reported above, except that the reaction was initiated with Cal+ added to the system 30 s after the mitochondria.
Under these conditions of excess Ca*+, oxygen utilization was stimulated and proceeded at a rapid rate until all the oxygen in the vessel was exhausted. The oxygen consumed was determined at each time point for which Ca2+ and 3-hydroxybutyrate were measured by subtracting the amount of oxygen consumed in a parallel experiment with no added Ca2+, giving a value of "extra oxygen consumed." "Extra 3-hydroxybutyrate uptake" was calculated as described above; "extra Cal+ uptake" was calculated by subtracting the value of 50 nmol/mg of protein obtained from Fig. 3 from each value of Ca2+ uptake. This value may not be very precise, due to variations between different mitochondrial preparations and to the small contribution of oxygen-utilizing Ca*+ uptake dependent on endogenous phosphate movements (see below). However, its magnitude was not critical since variation by 20% in either direction would alter the calculated Ca2+/w ratio only by about 0.2.
The extra uptake of Ca*+ and 3-hydroxybutyrate plotted against extra uptake of oxygen is presented in Fig. 4. The slopes of the lines plotted by the method of least squares and forced through the origin were calculated to be 3.8 for CaZ+ and 8.2 for 3-hydroxybutyrate.
Since succinate was the source of reducing equivalents, these values correspond to a Ca2+/w ratio of 1.9 and an associated 3-hydroxybutyrate/-ratio of 4.1. The extra oxygen uptake was also determined in the presence of 3-hydroxybutyrate and limiting amounts of Ca*+, in which case oxygen consumption returned to the basal rate before it was exhausted. Calculation of Caz+/-ratios under these conditions also yielded a value close to 2.0. Concentration Dependence of 3-HydroxybutyratelCa'+ Ratio-In order to assess whether 3-hydroxybutyrate could support loads of Ca2+ approaching those seen in the presence of phosphate and whether the stoichiometry of acid to Ca'+ was an invariant feature of extensive uptake of Ca*+ in the presence of 3-hydroxybutyrate, we investigated the dependence of the uptake on Ca*+ and acid concentration. Fig. 5 shows that, with a lo-min incubation, optimum uptake of both Ca*+ and 3-hydroxybutyrate was seen at about 300 to 400 nmol of Ca2+/mg of protein; virtually all the Ca*+ was accumulated at Ca2+ concentrations of less than 200 nmol/mg of protein. Fig. 6 shows a similar experiment in which 3-hydroxybutyrate concentration was varied; greatest uptake was observed between 15 and 30 mM acid. Also shown in Fig. 6 is the dependence of 3-hydroxybutyrate uptake on concentration in the absence of Ca2+; the fit of the points to the calculated line, assuming 3-hydroxybutyrate diffuses into and equilibrates with a matrix volume of 1 pl/mg of protein (see "Experimental Procedure"), is fairly good. Unlike the Ca*+ case, the uptake of 3-hydroxybutyrate did not approach completion at limiting For details see Fig. 2. CaCl, was added at different concentrations as shown; 3-hydroxybutyrate was 20 mM. The incubation was for 10 min at 23". acid concentrations.
Thus at 5 mM 3-hydroxybutyrate (2000 nmol/mg of protein), only 100 nmol of 3-hydroxybutyrate/mg of protein were accumulated. Assuming a matrix volume of I J/mg of protein, this corresponded to a 3-hydroxybutyrate gradient of 2O:l with the higher concentration within the mitochondria.
A similar gradient was calculated for each point at which 3-hydroxybutyrate was limiting, and may be explained by the limitation on the magnitude of the proton gradient which supported the 3-hydroxybutyrate uptake. Both Figs. 6 and 6 also show that accumulation of Ca2+ and R-hydroxybutyrate had a similar dependence on concentration; the decrease in steady state uptake at higher concentrations was attributed to membrane damage as discussed above.
The stoichiometry of the accumulation under conditions of limiting amounts of Ca2+ and 3-hydroxybutyrate is shown in Fig. 7, A and B respectively. In both cases a slope of I.7 was found. This is slightly lower than the value of 2.0 found in Fig.  3 and may be explained for Ca2+ by the decrease in nonenergized binding at low Caz+ concentrations, with a corresponding tendency for the intercept to decrease when Ca*+ is limiting. Brierley et al. (35) have shown that under some conditions respiration-dependent uptake of acetate and K+ may occur in the absence of other cation movements. Had a similar effect existed with 3-hydroxybutyrate in the present work the 3hydroxybutyrate/Ca*+ ratio would have been overestimated due to the replacement of Ca2+ by K+. Reducing the Ca*+ concentration should therefore have increased the proportion of the acid uptake dependent on K+ and raised the 3-hydroxybutyrate/Cal+ ratio. Fig. 7 shows that this did not occur; K+-dependent movements were therefore unimportant under our experimental conditions. Movement of Endogenous Acids-The cause of the Ca2+ uptake not associated with accumulation of 3-hydroxybutyrate seen as the intercept in Fig. 3

incubation
(i.e. after about 20 s, before centrifugation prevented further uptake) was 80.7 k 11.7 (SD.) nmol/mg of protein in the control, whereas with preincubation with N-ethylmaleimide this was reduced by over 50% to 37.0 + 4.2 (S.D.) nmol/mg of protein. Endogenous phosphate was therefore present at high enough activity to cause significant Ca2+ uptake in the absence of 3-hydroxybutyrate.
Although the uptake of phosphate in the presence of 20 mM 3-hydroxybutyrate would be greatly reduced it may nonetheless have made some contribution to the 3-hydroxybutyrate-independent uptake. Other factors involved may have been nonspecific binding of Ca*+ to the mitochondrial membranes and also the presence of dissolved CO, acting as a permeant acid (26). Cu2+ Uptake Supported by Other Monocarboxylic Acids-Since 3-hydroxybutyrate is oxidized in the absence of rotenone by rat liver mitochondria, the possibility remained that the effects observed were due to some specific interaction of 3-hydroxybutyrate with the mitochondria and not merely to its property of being a permeant weak acid. For this reason a number of other monocarboxylic acids were tested for their ability to support Ca*+ accumulation, and the stoichiometry of the uptake was measured in the same way as before. The results of such experiments are presented in Table I.
Acetate, propionate, and butyrate mimicked the, effects of 3-hydroxybutyrate with respect to the time course, extent, and stoichiometry of accumulation. 4-Hydroxybutyrate supported a much slower rate of uptake, which was incomplete at 8 min, and yielded a lower acid/Ca'+ ratio, presumably because endogenous ions were able to compete more effectively at lower rates of uptake. Glycolate was not taken up and did not support uptake of Ca2+, presumably because it is highly polar and does not pass the membrane. Phenylacetate supported only low levels of Ca2+ accumulation, although uptake was maximal at the first time point (i.e. after 20 s).
The extent of proton ejection caused by addition of Ca*+ to Conditions were ldentlcal with those of Fig. 2 except that :I-hydroxybutyrate was replaced by the various acids shown, at a concentration of 20 mM. Ca2+ was present at 400 nmol/mg of protein. The acid/Ca*+ ratio was calculated as described for Fig. 3. Initial rapid H+ ejection in the absence of added permeant acid was about 120 ng ions of H+/mg of protein. The presence of the added weak acid anions dimmished the control H+ ejection to the levels shown (see text). ' Caz+ uptake was still increasing linearly at 8 mm.
the medium 30 s after addition of mitochondria was measured and found to be small compared to the uptake of Ca2+ and the various acids, being about 60 ng ions of H+/mg of protein in the presence of 3-hydroxybutyrate compared to about 120 ng ions/mg of protein in the absence of a permeant acid. The table shows the ability of the acids tested to eliminate net accumulation of protons in the medium by transporting them into the matrix.
Those acids which supported Ca2+ uptake and were themselves readily taken up by the mitochondria (acetate, propionate, butyrate, 3-hydroxybutyrate) were most efficient at reducing proton production. 4-Hydroxybutyrate only partially eliminated acidification of the medium, consistent with its entry being restricted; glycolate could neither support uptake of Ca*+ nor reduce proton ejection, consistent with an inability to penetrate the mitochondrial membrane together with a proton. Phenylacetate reduced proton production but did not support high levels of Caz+ uptake, owing to its uncoupling action at this high concentration. The table also shows the pK' values for the acids; entry and support of Ca*+ uptake were not obviously related to pK'; other factors, particularly lipid solubility, presumably determine the differences between isomers such as 3-and 4-hydroxybutyrate.
It should be noted that the acid/Ca2+ ratio was the same for both the sodium salt (3-hydroxybutyrate) and the potassium salts (other acids), showing that the effects observed were independent of the accompanying monovalent cation.
Mitochondria from Malignant Cells-Mitochondria isolated from some malignant cells are able to accumulate Ca2+ and phosphate with particularly pronounced capacity to retain high loads ' (36-38). Uptake of Caz+ and 3-hydroxybutyrate by mitochondria isolated from AS-SOD ascites cells was investigated in experiments similar to those of Fig. 2