Attila Fonyó

Phosphate carrier of rat-liver mitochondria: Its role in phosphate outflow

Abstract It has been reported recently that inhibition of oxidative phosphorylation by some organic mercurial compounds was due to inhibition of phosphate entry into the mitochondria ( Tyler, 1968 ; Fonyo, 1967 ; Fonyo and Bessman, 1968) . Chappell and Crofts (1965) postulated earlier that phosphate penetrated the mitochondrial membrane via a carrier system. The possibility was therefore raised that the mercurials interacted with the phosphate carrier. In this paper evidence is presented that the outflow of phosphate from mitochondria is mediated by the same mercurial-sensitive system that permits phosphate entry.

Parallel measurement of oxoglutarate dehydrogenase activity and matrix free Ca2+ in fura-2-loaded heart mitochondria.

The entrapment of the Ca2+ ‐sensitive fluorescence indicators fura‐2 or quin2 in the matrix space of isolated heart mitochondria renders possible the direct monitoring of the matrix free Ca2+ ([Ca2+]m) [(1987) Biochem. J. 248, 609–613]. In this paper the correlation between the [Ca2+]m and the in situ activity of oxoglutarate dehydrogenase (OGDH) in fura‐2‐loaded mitochondria is shown. At the initial value of [Ca2+]m, 64 nM, which corresponded to 0.36 nmol/mg mitochondrial Ca content, the OGDH activity was 12% of the maximal. Half‐maximal and maximal activation were attained at 0.8 and 1.6 μM [Ca2+]m, respectively. The results indicate that an increase of the mitochondrial Ca content in the physiological range enhances the OGDH activity by means of elevation of [Ca2+]m.

Phosphate carrier of liver mitochondria: Two equivalent SH-groups in the carrier unit

Abstract Phosphate carrier activity of mitochondria that had become swollen during the aerobic accumulation of calcium acetate was measured indirectly by monitoring the turbidity changes. The exchange between extramitochondrial phosphate and intramitochondrial acetate was inhibited by SH-group binding reagents. Part of the SH-groups of the carrier could be blocked by mersalyl without loss of activity whereas liberation of the same groups restored carrier activity when all the other SH-groups were irreversibly blocked. A symmetrical structure of the carrier is proposed with two equivalent SH-groups; each of them is able in itself to maintain carrier function.

Is the mitochondrial Ca2+ uniporter a voltage-modulated transport pathway?

The influence of membrane potential (Δψ) on Ca2+ transport through the Ca2+ uniporter (UP) was investigated in fura‐2‐loaded rat heart mitochondria at physiologically relevant‐submicromolar‐external [Ca2+]. In the absence of Δψ the UP could not mediate Ca2+ uptake even when an 8‐fold external (∼500 nM) to internal (∼60 nM) [Ca2+] gradient was present and charge compensation was provided by acetate and the protonophore, CCCP. A small (∼−120 mV) and transient Δψ (generated by valinomycin) resulted in a rise in matrix [Ca2+] only when external [Ca2+] exceeded 150 nM. At physiologically high (∼−180 mV) and stable Δψ this threshold value for Ca2+ uptake dropped to 15 nM. The results indicate that (1) at physiological [Ca2+]m Δψ in addition to being a component of ΔμCa2+ seems to be necessary for providing a transport‐competent conformation for the UP; and (2) below a threshold [Ca2+]m the UP cannot operate even in the presence of a high electric driving force.

The Ba2+ sensitivity of the Na+-induced Ca2+ efflux in heart mitochondria: the site of inhibitory action

The Na+-induced Ca2+ release from rat heart mitochondria was measured in the presence of Ruthenium red. Ba2+ effectively inhibited the Na+-induced Ca2+ release. At 10 mM Na+ 50% inhibition was reached by 1.51 +/- 0.48 (S.D., n = 8) microM Ba2+ in the presence of 0.1 mg/ml albumin and by 0.87 +/- 0.25 (S.D., n = 3) microM Ba2+ without albumin. In order to inhibit, it was not required that Ba2+ ions enter the matrix. 140Ba2+ was not accumulated in the mitochondrial matrix space; further, in contrast to liver mitochondria, Ba2+ inhibition was immediate. The Na+-induced Ca2+ release was inhibited by Ba2+ non-competitively, with respect of the extramitochondrial Na+. The double inhibitor titration of the Na+-Ca2+ exchanger with Ba2+ in the presence and absence of extramitochondrial Ca2+ revealed that the exchanger possesses a common binding site for extramitochondrial Ca2+ and Ba2+, presumably the regulatory binding site of the Na+-Ca2+ exchanger, which was described by Hayat and Crompton (Biochem. J. 202 (1982) 509-518). All these observations indicate that Ba2+ acts at the cytoplasmic surface of the inner mitochondrial membrane. The inhibitory properties of Ba2+ on the Na+-dependent Ca2+ release in heart mitochondria are basically different from those found on Na+-independent Ca2+ release in liver mitochondria (Lukács, G.L. and Fonyó, A. (1985) Biochim. Biophys. Acta 809, 160-166).

Competitive inhibition of valinomycin-induced K+-transport by Mg2+-ions in liver mitochondria.

Mitochondria are almost impermeable for K+-ions. This restriction of K’-ion permeability is apparently essential for mitochondrial function. As has been shown earlier the limitation of alkali-cation permeability depends on the presence of Mg2+-ions. Simple chelation of Mg2+ by EDTA increased the K*-ion permeability of isolated liver mitochondria [l-3] . EDTA increased also Na+-ion permeability [4] . Addition of Mg2+-ions restored the permeability for alkali cations to the original, low level. Very recently the divalent-cation ionophore, A 23 187 was used in combination with EDTA to deplete the mitochondria of their endogenous Mg2+-ion content: as a result the permeability for K+-ions was greatly increased [S] . All these effects were taken as evidence that a postulated endogenous K+-H* exchange system is operative in the absence of Mg2+-ions and is inactivated in its presence [5]. It was supposed that either the same or a similar exchanger is responsible for the transport of Na2+-ions [4] . The mechanism by which Mg’+-ions would change the membrane permeability was left open. One of the possibilities is that Mg2+-ions change the physical structure of the membrane, its inner fluidity, in a way that the intra-membrane mobility of the exchanger decreases. This hypothesis could be tested by investigating the effect of Mg2+-ions on transport catalyzed by exogenous carriers. The K+-ion permeability of membranes is radically increased by the ionophore, valinomycin (see [6 ] for

Na+/H+ exchange in mitochondria as monitored by BCECF fluorescence

The recently developed method of loading isolated heart mitochondria with the fluorescent pH indicator, BCECF, was applied to monitor the Na+ 0/H+ i exchange process from the matrix side of the membrane. The Na+‐induced changes in the pH of the matrix (pHm) showed that: (i) the Na+ 0/H+ i exchange followed Michaelis‐Menten kinetics with respect to external Na+ with a K m of approx. 20 mM; (ii) in contrast to this, the dependence of the exchange rate on the matrix [H+] did not obey the Michaelian model. No Na+‐induced alkalinization occurred above a pHm of 7.45±0.09 (n=4). Below this value the reciprocal of the transport rate and that of the matrix [H+] deviated upwardly from the straight line. The results suggest that internal H+ might exert allosteric control on the mitochondrial Na+/H+ exchange process.

Transport properties and inhibitor sensitivity of isolated and reconstituted porin differ from those of intact mitochondria

The pore-forming protein porin has been isolated from rat heart mitochondria and reconstituted in phospholipid vesicles of different composition. Rapid release of anions, cations and non-charged molecules has been demonstrated from the proteoliposomes but not from the protein-free liposomes. In spite of its higher molecular mass and charges, the movement of ATP was almost as fast as that of inorganic phosphate. Polyanion (1:2:3 copolymer of methacrylate/maleate/styrene), a potent inhibitor of porin residing in the mitochondrial contact sites decreased the solute movements but did not completely block any of the investigated transport processes (phosphate, chloride, ATP). Alterations of the lipid environment had significant effect: an increase in the proportion of soybean phospholipids to egg yolk phospholipids resulted in a decrease in the amount of transported substance but did not fully inhibit the ion movements. It is concluded that the transport properties of porin reconstituted in artificial phospholipid membranes are different from the characteristics of porin prevailing in the mitochondrial contact sites and additional regulatory factors are suggested to be effective in the intact mitochondria.

Ba2+ ions inhibit the release of Ca2+ ions from rat liver mitochondria

The release of Ca2+ from respiring rat liver mitochondria following the addition of either ruthenium red or an uncoupler was measured by a Ca2+-selective electrode or by 45Ca2+ technique. Ba2+ ions are asymmetric inhibitors of both Ca2+ release processes. Ba2+ ions in a concentration of 75 microM inhibited the ruthenium red and the uncoupler induced Ca2+ release by 80% and 50%, respectively. For the inhibition, it was necessary that Ba2+ ions entered the matrix space: Ba2+ ions did not cause any inhibition of Ca2+ release if addition of either ruthenium red or the uncoupler preceded that of Ba2+. The time required for the development of the inhibition of the Ca2+ release and the time course of 140Ba2+ uptake ran in parallel. Ba2+ accumulation is mediated through the Ca2+ uniporter as 140Ba2+ uptake was competitively inhibited by extramitochondrial Ca2+ and prevented by ruthenium red. Due to the inhibition of the ruthenium red insensitive Ca2+ release, Ba2+ shifted the steady-state extramitochondrial Ca2+ concentration to a lower value. Ba2+ is potentially a useful tool to study mitochondrial Ca2+ transport.

Intramitochondrial phosphate is the source of protons in the response of liver mitochondria to cations

We have reported that after inhibition of phosphate transport in mitochondria, their respiratory response to divalent cations (Ca’+, Sr2’) was proportional to the intramitochondrial phosphate content [l] . A similar conclusion has been reached in [2] . We suggested that intramitochondrial phosphate was the source of protons for the proton pump linked to the respiratory chain. The interpretation of these results raises two further questions: 1. Is the dependence of the respiratory response on intramitochondrial phosphate specific for c’ivalent cations which have a specific porter system in mitodria? The importance of this question was stressed by the suggestion [3,4] concerning a mersalylinsensitive phosphate transport linked to uptake of divalent cations. 2. Is there experimental support for the suggested relationship between intramitochondrial phosphate and proton ejection? We have found that the temporary stimulation of respiration following the addition of K’ plus valinomycin was parallel to the intramitochondrial phosphate content when the latter was changed experimentally. A similar parallelism between intramitochondrial phosphate and proton ejection was also present. This supports the view that intramitochondrial phosphate is the proton donor to the respiratory chain-linked proton pump after phosphate transport inhibition in mitochondria.