Comparative partial purification of the active dicarboxylate transport system of rat liver, kidney and heart mitochondria

doi:10.1016/0006-291X(85)90934-9

Biochemical and Biophysical Research Communications

Volume 133, Issue 2, 17 December 1985, Pages 498-504

https://doi.org/10.1016/0006-291X(85)90934-9 Get rights and content

Abstract

Hydroxylapatite chromatography of Triton-extracted inner-membrane proteins from rat liver mitochondria allowed a ten-fold purification of the dicarboxylate carrier. The purified system, reconstituted into liposomes, displayed all the properties of the dicarboxylate carrier and mediated malonate-malate and malonate-phosphate exchanges. Six protein bands of M_r ranging from 27,000 to 34,000 could be resolved by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The purification of the dicarboxylate carriers of liver, kidney and heart mitochondria were carried out by this method and their properties were compared with respect to transport activity and electrophoresis patterns. Our results demonstrate that the dicarboxylate carrier of rat mitochondria can be obtained in an advanced state of purification and with a high specific activity.

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These results, together with substrate-dependent reversibility of NADH/H2O2 production in permeabilized mitochondria, suggest that the actual concentration of substrates/products in the vicinity of reversible matrix enzymes have an important role. Cardiac mitochondria reportedly exhibit dicarboxylate carrier activity that is six times lower compared with liver mitochondria [7]. In addition to relatively low carrier activity, glutamate uptake through glutamate/aspartate antiporter is electrogenic and requires membrane potential [8,9].
Reactive oxygen species (ROS) production by isolated complex I is steeply dependent on the NADH/NAD⁺ ratio. We used alamethicin-permeabilized mitochondria to study the substrate-dependence of matrix NADH and ROS production when complex I is inhibited by piericidin or rotenone. When complex I was inhibited in the presence of malate/glutamate, membrane permeabilization accelerated O₂ consumption and ROS production due to a rapid increase in NADH generation that was not limited by matrix NAD(H) efflux. In the presence of inhibitor, both malate and glutamate were required to generate a high enough NADH/NAD⁺ ratio to support ROS production through the coordinated activity of malate dehydrogenase (MDH) and aspartate aminotransferase (AST). With malate and glutamate present, the rate of ROS production was closely related to local NADH generation, whereas in the absence of substrates, ROS production was accelerated by increase in added [NADH]. With malate alone, oxaloacetate accumulation limited NADH production by MDH unless glutamate was also added to promote oxaloacetate removal via AST. α-ketoglutarate (KG) as well as AST inhibition also reversed NADH generation and inhibited ROS production. If malate and glutamate were provided before rather than after piericidin or rotenone, ROS generation was markedly reduced due to time-dependent efflux of CoA. CoA depletion decreased KG oxidation by α-ketoglutarate dehydrogenase (KGDH), such that the resulting increase in [KG] inhibited oxaloacetate removal by AST and NADH generation by MDH. These findings were largely obscured in intact mitochondria due to robust H₂O₂ scavenging and limited ability to control substrate concentrations in the matrix. We conclude that in mitochondria with inhibited complex I, malate/glutamate-stimulated ROS generation depends strongly on oxaloacetate removal and on the ability of KGDH to oxidize KG generated by AST.
Evaluation of molecularity of rate-limiting step of pore formation by antimicrobial peptides studied using mitochondria as a biosensor
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The standard RLM concentration in the oximetric cell was 0.25 mg/ml except for experiments on determination of the partition coefficient (Kp) for distribution of TAM between RLM and the incubation medium. SMP were obtained by the simplified method of Saint-Macary and Foucher (1985). Briefly, the RLM preparation was placed in hypotonic medium containing 4 mM Tris (pH 7.4) and 2 mM EDTA for 20 min at 0 °C, and after addition of sucrose to 250 mM the mitoplasts were pelleted by centrifugation at 6000g for 20 min.
Toxic agents, derived from bee or hornet venoms and from fungi – melittin, mastoparan, and alamethicin are able to permeabilize biological membranes. We studied the initial steps of pore formation by these peptides in rat liver mitochondria preparations (RLM) generating transmembrane potential (ΔΨ). RLM has been used as a potassium transmembrane current (PTC) sensor. The PTC induced in RLM depends linearly on the degree of steady-state activation of RLM respiration. The concentration order of such activation by melittin in a “potassium” incubation medium containing 6 mM Mg²⁺ was 2.01 ± 0.15. In the case of mastoparan, the reaction order was 1.83 ± 0.23. The first steady-state phase of activation of RLM respiration by alamethicin was not detected in “Tris” incubation medium; it appeared only after addition of KCl. The order of the reaction limiting such activation was 1.92 ± 0.07. It is suggested that PTC in this phase is determined by the channels with the lowest degree of oligomerization formed by “dimers”. The ratio of equally active membrane concentrations of peptides obviously reflects the ratio of average lifetimes (ALT) for corresponding “dimers” (alamethicin and melittin, 38.5; mastoparan and melittin, 0.32). It is concluded that the results of this investigation may be useful for comparative testing of perspective pharmaceuticals.
A bioenergetic model of the mitochondrial population undergoing permeability transition
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Mitochondria from specific tissue types are phenotypically different and contain different amounts of innermembrane proteins, matrix proteins and lipid types optimized to support their designated function. For example, cardiac mitochondria are reported to possess a less active dicarboxylate carrier than the liver mitochondria (Saint-Macary and Foucher, 1985). This carrier is responsible for the exchange of primarily Pi, malate and succinate.
Mitochondrial permeability transition (MPT) is a highly regulated complex phenomenon that is a type of ischemia/reperfusion injury that can lead to cell death and ultimately organ dysfunction. A novel population transition and detailed permeability transition pore regulation model were integrated with an existing bioenergetics model to describe MPT induction under a variety of conditions. The framework of the MPT induction model includes the potential states of the mitochondria (aggregated, orthodox and post-transition), their transitions from one state to another as well as their interaction with the extra-mitochondrial environment. The model encodes the three basic necessary conditions for MPT: a high calcium load, alkaline matrix pH and circumstances which favor de-energization. The MPT induction model was able to reproduce the expected bioenergetic trends observed in a population of mitochondria subjected to conditions that favor MPT. The model was corroborated and used to predict that MPT in an acidic environment is mitigated by an increase in activity of the mitochondrial potassium/hydrogen exchanger. The model was also used to present the beneficial impact of reducing the duration mitochondria spend in the orthodox state on preserving the extra-mitochondrial ATP levels. The model serves as a tool for investigators to use to understand the MPT induction phenomenon, explore alternative hypotheses for PTP regulation, as well as identify endogenous pharmacological targets and evaluate potential therapeutics for MPT mitigation.
Cardiolipin and mitochondrial carriers
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Citation Excerpt :
The isolation of the tricarboxylate carrier from liver mitochondria profited early from the CL interference [25] but it took some time to elucidate the intricacies for separating the tricarboxylate carrier [26]. Isolation of the dicarboxylate carrier from liver mitochondria also involved the use of CL for increasing the yield [27,28–30]. Similarly, the carrier for oxoglutarate isolated from heart [31] and from liver mitochondria [29] depended on the addition of CL for their isolation.
Members of the mitochondrial carrier family interact with cardiolipin (CL) as evident from a variety of functional and structural effects. CL stabilises carrier proteins on isolation with detergents, with the P_i carrier as the prime example. CL is required for transport in reconstituted vesicles, prime examples are the P_i- and ADP/ATP carrier (AAC). CL binds to the AAC in a graded manner; 6 CL/AAC dimer bind tightly as measured on the ³¹P NMR time scale. 2 additional CL/dimer bind reversibly and a fast exchanging envelope of phospholipids includes CL as measured on the ESR time scale. In the crystal structure of the CAT–AAC complex 3 CL bind to the periphery of the AAC in a three-fold pseudo-symmetry. The binding of CL is implicated to contribute lowering the high transition energy barriers in the AAC. Para-functions of the AAC, as in the mitochondrial pore transition (MPT) and in cell death are linked to the CL binding of the AAC. Ca⁺⁺ or oxidants can sequester or destroy AAC bound CL, rendering AAC labile, allowing pore formation and degradation. Thus AAC, by being vital for energy transfer, constitutes an Achilles heel in the eukaryotic cell. AAC together with CL is also engaged in respiratory supercomplexes. Different from AAC the similarly structured uncoupling protein (UCP1) has no tightly bound CL, but CL addition lowers affinity of the inhibitory nucleotide binding that may contribute to the physiological regulation of the uncoupling activity by ATP.
Characterization, purification and properties of the yeast mitochondrial dicarboxylate carrier (Saccharomyces cerevisiae)
1996, Biochimie
The dicarboxylate carrier has been characterized and purified from mitochondria of wild strain Saccharomyces cerevisiae. The mitochondria were solubilized with Triton X-100 and the detergent extract was chromatographed on hydroxylapatite. SDS-PAGE of the hydroxylapatite pass-through showed five protein bands with M_rs ranging from 28 000 to 35 000, by silver nitrate staining. The n-butylmalonate-sensitive succinate_out/malate_in exchange activity of the hydroxylapatite pass-through reconstituted into liposomes, was increased nine-fold with respect to the activity of the Triton X-100 extract. The exchange activity was inhibited by p-chloromercuriphenylsulfonate (PMPS), 4,4′diisothiocyanostilbene-2,2′-disulfonate (DIDS) and pyridoxal-phosphate, suggesting that one or more thiol groups and basic residues are implicated in the binding mechanism. The purification of the carrier was achieved by affinity chromatography on Sepharose-immobilized malate dehydrogenase. The purified protein presented the same properties as the dicarboxylate carrier in native mitochondria and displayed a single protein band with an M_r of 28 000 as determined by SDS-PAGE. The specific activity of the purified carrier showed a 53-fold increase compared to that of the initial material. The K_m for the reconstituted exchange was 2 mM for succinate with a V of 1.5 μmol min⁻¹ mg⁻¹ protein at 22°C. The high purification state achieved for the yeast dicarboxylate carrier should allow the study of its molecular properties.
Purification of the rat-liver mitochondrial dicarboxylate carrier by affinity chromatography on immobilized malate dehydrogenase
1994, BBA - Biomembranes
The dicarboxylate carrier of rat-liver mitochondria, extracted by Triton X-100 and partially purified by hydroxylapatite chromatography, was retained by malate dehydrogenase immobilized on Sepharose gel, and eluted with 0.4 M NaCl. SDS-polyacrylamide gel electrophoresis of the eluate showed a predominant peptide band with an M_r of 28 000. The purified protein, incorporated into liposomes, mediated a butylmalonate sensitive malonate_out/malate_in exchange that was inhibited by $p- chloromercuriphenylsulfonate$ . Sulfate, malate and phosphate decreased the rate of exchange. The highly purified protein displayed all the properties of the dicarboxylate carrier. Moreover, the results suggest a possible functional interaction between mitochondrial carrier protein and malate dehydrogenase.

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Comparative partial purification of the active dicarboxylate transport system of rat liver, kidney and heart mitochondria

Abstract

FEBS Lett

Biochim. Biophys. Acta

FEBS Lett

FEBS Lett

FEBS Lett

FEBS Lett

Biochem. Biophys. Res. Commun