Bernard Foucher

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

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 Mr 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.

Purification of the rat-liver mitochondrial dicarboxylate carrier by affinity chromatography on immobilized malate dehydrogenase

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.

Reconstitution of the dicarboxylate exchange activity by incorporation into liposomes of a Triton-extract of mitochondrial rat-liver inner membranes

The exchange between external [14C] malonate and internal malate or phosphate was reconstituted in liposomes prepared by incorporation of a Triton-extract of mitochondrial rat-liver inner membranes. The conditions of transport were investigated and the kinetic parameters of malonate-malate and malonate-phosphate exchanges were determined. The exchange was sensitive to butylmalonate and to organomercurials. Sulfate and phosphate decreased the rate of malonate-malate exchange and phosphate inhibition was found to be competitive. This report demonstrates the possibility to reconstitute a functional dicarboxylate transport and to use the system for further purification and for studies at the molecular level.

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex.

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.

Dicarboxylate transport in the inner membrane matrix fraction obtained from rat liver mitochondria

Dicarboxylate transport was studied in the inner membrane matrix fraction (mitoplasts) and compared to that in intact rat-liver mitochondria from which the former was obtained. It is concluded that, kinetics of dicarboxylate exchange measured in mitoplasts, are very similar to those observed with mitochondria. These results would indicate that the preparation technique preserves the integrity of the inner membrane and that neither the outer membrane nor the components of the peripheral space affect these results.

Influence of adenine nucleotides on oligomycin inhibition of energy-transducing reactions in intact rat-liver mitochondria

The inhibitory effect of oligomycin was investigated in intact mitochondria through oxidative phosphorylation and uncoupler induced ATPase activity. Results show that oligomycin inhibition curves can be either sigmoidal or hyperbolic depending on experimental conditions and chiefly on the metabolic state of mitochondria with regard to the distribution of mitochondrial endogenous adenine-nucleotides. Active respiration and uncoupler-induced ATPse activity produce sigmoidal titration curves for a high initial ATP : ADP ratio and hyperbolic curves for a low ATP : ADP ratio. Time-dependent inhibitions are observed for the two reactions. The maximal inhibitory action for low concentrations of the inhibitor is delayed by the initial presence of ATP or the possibility of generating from inorganic phosphate before adding oligomycin. Results presented here show that the initial adenine-nucleotide distribution is important for oligomycin sensitivity of energy-linked reactions. Although a limited conformational change of the oligomycin-sensitivity to the inhibitor, it is more likely that a gross structural change of the inner membrane induced by adenine-nucleotides modifies membrane permeability to oligomycin.

Neurospora crassa pyruvate dehydrogenase complex: component characterization, catalytic properties and location of translation

We propose a simplified procedure for the purification of the Neurospora crassa pyruvate dehydrogenase complex. The purified complex showed four protein bands with apparent Mr values of 53,400, 52,900, 49,000 and 36,900 upon SDS-polyacrylamide gel electrophoresis. Components, E2 and E3, of N. crassa pyruvate dehydrogenase complex were identified, respectively, as polypeptides 49,000 and 53,400. It can be deduced that component E1 is constituted of two subunits with Mr values of 52,900 and 36,900. The Km values towards different substrates and the optimal pH and temperature were determined. The protein kinase activity associated with the core enzyme was present in our most highly purified preparations. It was demonstrated that all the protein components of the complex are synthesized under the control of the nuclear genome.

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

Binding of malonate to the inner membrane of rat liver mitochondria

Abstract The binding of malonate to the external face of the mitochondrial inner membrane has been investigated by using mitoplasts and on the opposite face, by using inside-out oriented vesicles prepared from sonicated mitoplasts. The external face of the inner membrane displays a single class of binding sites whereas two classes are observed in vesicles. In trypsin treated vesicles, only high-affinity sites are evidenced. The disappearance of the low affinity sites is correlatively related with the loss of succinate dehydrogenase activity. Both high-affinity sites of mitoplasts and vesicles are sensitive to the presence of malate; they are also masked by 2-butylmalonate, phosphate, citrate and mercurials. Our results suggest that the internal and external high-affinity sites of the inner membrane are involved in the dicarboxylate transport system.

Neurospora crassa α-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties

A method is proposed for the purification of the Neurospora crassa α-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-poly-acrylamide gel electrophoresis as E3, E1 and E2 with respective Mr values of 54000, 53 000 and 49000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca2+ in particular was investigated. It was shown that Ca2+ diminishes the Km value of the N. crassa α-ketoglutarate dehydrogenase complex for α-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.