Ernesto Carafoli

Ca2+ metabolism in yeast cells and mitochondria

Abstract 1. 1. Ca 2+ in the culture medium stimulates only slightly the growth and respiration of Saccharomyces cerevisiae . 2. 2. Neither energy-linked Ca 2+ transport nor high-affinity Ca 2+ binding occur in mitochondria isolated from Saccharomyces cerevisiae or from Torulopsis utilis . 3. 3. Metabolism-independent, low-affinity binding of Ca 2+ does, however, occur in mitochondria isolated from both yeasts. The concentration of these sites varies between 40 and 50 nmoles per mg of mitochondrial protein. Their affinity for Ca 2+ is rather low ( K m = 10–20 μ M). 4. 4. Mitochondria of Saccharomyces cerevisiae contain about 10 nmoles of endogenous Ca 2+ per mg of mitochondrial protein, which is bound or sequestered in a very stable manner. 5. 5. The respiration of mitochondria from Saccharomyces cerevisiae is stimulated by valinomycin in the presence of KCl, suggesting that energy-linked transport of K + occurs. 6. 6. The results show that energy-linked Ca 2+ transport is not a universal attribute of intact mitochondria from all species. Since the yeast cells lack high-affinity Ca 2+ binding capacity, they may lack a specific Ca 2+ carrier. The basic energy-dependent cation pump may however be present in these mitochondria, since they can transport K + in the presence of valinomycin.

The interaction of La3+ with mitochondria in relation to respiration-coupled Ca2+ transport

Abstract La 3+ inhibits the respiration-dependent accumulation of Ca 2+ by rat liver mitochondria when added in very small amounts (0.1–l.0 nmole per mg protein). However, La 3+ itself does not activate respiration. With the use of 140 La 3+ it was found that La 3+ is very rapidly bound to rat liver mitochondria in a respiration-independent process accompanied by loss of H + to the medium. When both La 3+ and Ca 2+ are added to mitochondria simultaneously, most of the La 3+ but little Ca 2+ are bound. La 3+ added to mitochondria previously loaded with Ca 2+ is tightly bound without discharge of Ca 2+ . Conversely, when Ca 2+ is added to La 3+ -loaded mitochondria it is not bound nor is the La 3+ discharged. La 3+ inhibits both high-affinity and low-affinity respiration-independent Ca 2+ binding. Isotopic experiments showed that La 3+ is, in fact, bound to the same high-affinity sites as Ca 2+ , in both intact mitochondria and in mitochondrial extracts. It is concluded (1) that La 3+ binds to and inhibits the Ca 2+ carrier; (2) that La 3+ is not transported by the Ca 2+ carrier; and (3) that La 3+ is, in addition, bound to a large number of external sites on mitochondria for which Ca 2+ is not a strong competitor.

Active accumulation of Sr2+ by rat-liver mitochondria I. General features

Abstract 1. 1. Sr 2+ is rapidly accumulated from the suspending medium by respiring rat-liver mitochondria in an energy-linked process. The accumulation proceeds in two phases: a rapid phase, requiring only seconds, and a slow phase, requiring several min. Almost half the Sr 2+ uptake occurs in the rapid phase, which requires respiration in the presence of phosphate, but not adenosine 5′-triphosphate or Mg 2+ . The second phase requires respiration in the presence of adenosine 5′-triphosphate, inorganic phosphate and Mg 2+ . Maximum rates of Sr 2+ uptake occur at a Sr 2+ concentration of 2 mM; the maximum amount accumulated is about 2.5 μmoles Sr 2+ per mg mitochondrial protein. Oligomycin inhibits neither phase of respiration-supported Sr 2+ uptake; 2,4-dinitrophenol (DNP) inhibits both. 2. 2. Sr 2+ uptake may also be supported by adenosine 5′-triphosphate alone, even when respiration is inhibited by CN − ; in this case two phases of Sr 2+ uptake are also observed. However the rapid phase of adenosine 5′-triphosphate-supported Sr 2+ uptake is not inhibited by oligomycin. 3. 3. Sr 2+ uptake in both phases is accompanied by uptake of phosphate; the Sr/inorganic phosphate accumulation ratio is 1.2–1.4. The rapid phase of Sr 2+ uptake is stimulated by K + , the slow phase is inhibited by K + .

Active accumulation of Sr2+ by rat-liver mitochondria III. Stimulation of respiration by Sr2+ and its stoichiometry

Abstract 1. 1. Addition of Sr 2+ to rat-liver mitochondria suspended in a medium of respiratory substrate, Mg 2+ , and phosphate causes a large stimulation of respiration, the duration of which is proportional to the Sr 2+ added. The rate of respiration then returns to the original level. During the period of respiratory stimulations Sr 2+ is accumulated by the mitochondria; when the Sr 2+ of the medium is exhausted, respiration returns to the original low rate. Tests with β-hydroxybutyrate and succinate show that between 1.8 and 2.0 ions of Sr 2+ are accumulated as a pair of electrons traverses each of the three energy-coupling sites of the respiratory chain. 1 atom of extra oxygen was taken up during accumulation of 3.8 moles of Sr 2+ when succinate was the substrate, and 5.5 moles of Sr 2+ when β-hydroxybutyrate was the substrate. Succinate oxidation is stimulated to higher rates by addition of Sr 2+ than is β-hydroxybutyrate oxidation. Accumulation of phosphate accompanies accumulation of Sr 2+ . 2. 2. When phosphate is not present in the medium, Sr 2+ also stimulates respiration, but in this case the return to the inhibited state takes place only with concentrations of Sr 2+ lower than 400–500 μM. The respiration continues indefinitely at the stimulated rate when higher concentrations of Sr 2+ are added. The accumulation of Sr 2+ in the absence of phosphate is much smaller than in its presence. Phosphate is therefore not required for stimulation of oxygen uptake but only for accumulation of large amounts of Sr 2+ ; when the latter is exhausted, the oxygen uptake ceases.

ACTIVE ACCUMULATION OF SR2+ BY RAT-LIVER MITOCHONDRIA. II. COMPETITION BETWEEN CA2+ AND SR2+.

Abstract Ca2+ inhibits the respiration-linked accumulation of Sr2+ in a competitive manner. Both ions may be accumulated simultaneously from the medium, the relative amounts of each being determined by the competition. The inhibiting action of Ca2+ is primarily on the rapid phase of Sr2+ uptake. When the ion accumulation is supported by adenosine 5′-triphosphate instead of respiration, the inhibiting effect of Ca2+ on Sr2+ uptake is much less pronounced. The release of both Sr2+ and Ca2+ from “loaded” mitochondria is promoted by 2,4-dinitrophenol.

The effect of Sr2+ on swelling and ATP-linked contraction of mitochondria

Abstract 1. 1. Although Sr 2+ and Ca 2+ are actively accumulated by rat-liver mitochondria during respiration by a common mechanism, Ca 2+ is a very potent swelling agent at low concentrations, whereas Sr 2+ shows no swelling activity. In fact, Sr 2+ protects mitochondria against spontaneous swelling or that induced by Ca 2+ , thyroxine, oleate, or phosphate. The protective action of Sr 2+ in some respects resembles that of bovine serum albumin, and in other respects that of Mg 2+ . When Sr 2+ protects mitochondria against swelling caused by phosphate, some Sr 2+ is accumulated from the medium. 2. 2. Sr 2+ supports maximum contraction of swollen mitochondria induced by ATP. Sr 2+ may completely replace both Mg 2+ and bovine serum albumin in supporting ATP-linked reversal of Ca 2+ -induced swelling. Sr 2+ also prevents reswelling of ATP-contracted mitochondria. When Sr 2+ is added to Ca 2+ -swollen mitochondria together with ATP, contraction is accompanied by accumulation of Sr 2+ , and by inhibition of Ca 2+ accumulation; no reswelling then occurs. The production of U factor or free fatty acids, which is accelerated by Ca 2+ , is not influenced by Sr 2+ . However, Sr 2+ does appear to block the normal swelling action of free fatty acids. Sr 2+ thus differs sharply from Ca 2+ in its action on the structural state of mitochondria, although active uptake of both ions shows the same stoichiometric relationship to respiration. 3. 3. Sr 2+ depresses the resting and the Ca 2+ -stimulated ATPase activity of rat-liver mitochondria. It also inhibits the Mg 2+ -stimulated ATPase activity of aged rat-liver-mitochondrial preparations. The 32 P i -ATP exchange activity was also found to be very markedly inhibited by Sr 2+ .

Energy-coupling in mitochondria during resting or state 4 respiration☆

Abstract The resting or State 4 respiration of freshly prepared, intact rat liver mitochondria can support energy-linked accumulation of Ca++ or Sr++ or the oxidative phosphorylation of ADP. However, under these conditions the coupling efficiency is relatively low; about 10% maximal for Ca++, about 20% for Sr++ accumulation, and about 12% maximal for oxidative phosphorylation of ADP.

On the maximum stoichiometry of energy-linked Ca++ accumulation during electron transport in rat liver mitochondria

Abstract Several recent investigations have shown that approximately 1.7 – 2.0 molecules of divalent cation (Ca++, Sr++, or Mn++) may be accumulated by isolated mitochondria coupled to the passage of a pair of electrons through each energy-conserving site of the respiratory chain ( Rossi and Lehninger, 1963 , Rossi and Lehninger, 1964 ; Chappell, Cohn, and Greville, 1963 ; Carafoli, Weiland, and Lehninger, 1965 ; Carafoli, 1965 ; Chance, 1963 , Chance, 1965 ). Most reported measurements of the Ca++:∼ stoichiometry have been carried out with inorganic phosphate in the medium; under such conditions phosphate is also accumulated. However, limited amounts of divalent cations may also be accumulated in the absence of phosphate, with apparently similar stoichiometry with oxygen uptake ( Rossi and Lehninger, 1964 ; Chappell et al. , 1963 ; Carafoli, 1965 ). This communication describes two sets of conditions under which Ca++:∼ accumulation ratios over twice as high as those previously reported may be observed. When the concentration of certain neutral salts in the medium, such as NaCl or KCl, is increased to 240 mM or higher, with pH held constant at 7.4, the Ca++:∼ ratio increases to values exceeding 4.0. When the pH is raised from 7.0 to 8.0, with the NaCl concentration held constant at 80 mM, the Ca++:∼ ratio increases to as high as 7.0. In the presence of phosphate, however, the Ca++:∼ ratio remains at 2.0, regardless of pH or salt concentration.

K+-dependent rebounds and oscillations in respiration-linked movements of Ca++ and H+ in rat liver mitochondria

Abstract This communication reports conditions in which respiration-linked uptake of Ca ++ by rat liver mitochondria (cf. Rossi and Lehninger, 1964 ; Chance, 1965 ) and the accompanying ejection of H + ( Sawis, 1963 ; Drahota et al., 1965 ; Chappell and Crofts, 1965 ) undergo striking K + -dependent rebound and oscillatory phenomena, without concomitant fluctuations in oxygen uptake. The findings provide further evidence that the stoichiometry between Ca ++ uptake and electron transport may vary considerably under different conditions ( Carafoli, Gamble, and Lehninger, 1965 ) and also suggest the existence of a feed-back relationship between active Ca ++ uptake and the Ca ++ efflux rate.

Active Ion Transport by Mitochondria

The ability of isolated mitochondria to accumulate very large amounts of Ca++ by an active transport mechanism was discovered by V a s i n g t o n and M u r p h y in 1961 [1]. The accumulation process was found to be blocked by respiratory inhibitors and by uncouplers of oxidative phosphorylation, and thus was completely dependent on electron transport and the energy coupling mechanism of oxidative phosphorylation. Significantly, oligomycin does not inhibit Ca++ uptake under these conditions. Later it was found that during the accumulation of Ca++, inorganic phosphate disappears from the medium and also accumulates in mitochondria in amounts that are stoichiometrically related to the Ca++; the molar ratio with which Ca++ and phosphate are accumulated is almost exactly that of Ca++ hydroxyapatite. The amounts of Ca++ and phosphate that accumulate may exceed 200 times the normal mitochondrial content; such accumulation is called “massive loading”. The accumulation of Ca++ and phosphate can also be driven by ATP hydrolysis, and in this case it is not sensitive to respiratory inhibitors, but is inhibited by uncouplers of oxidative phosphorylation and by oligomycin. Ca++ uptake is evidently associated with the dinitrophenolsensitive formation of the first high energy intermediate formed during electron transport, before the point of action of oligomycin.