Purification and characterization of the reconstitutively active adenine nucleotide carrier from maize mitochondria.

The adenine nucleotide carrier from maize (Zea mays L. cv B 73) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. Sodium dodecyl sulfate-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 32 kD. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5'-phosphate-sensitive ATP/ATP exchange. It was purified 168-fold with a recovery of 60% and a protein yield of 0.25% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ADP, ATP, GDP, and GTP, and was inhibited by atractyloside, bongkrekate, phenylisothiocianate, pyridoxal 5'-phosphate, and mersalyl (but not N-ethylmaleimide). Maximum initial velocity of the reconstituted ATP/ATP exchange was determined to be 2.2 mumol min-1 mg-1 protein at 25 degrees C. The half-saturation constants and the corresponding inhibition constants were 17 microM for ATP, 26 microM for ADP, 59 microM for GTP, and 125 microM for GDP. The activation energy of the ATP/ATP exchange was 48 kilojoule/mol between 0 and 15 degrees C, and 22 kilojoule/mol between 15 and 35 degrees C. Partial amino acid sequences showed that the purified protein was the product of the ANT-G1 gene sequenced previously (B. Bathgate, A. Baker, C.J. Leaver [1989] Eur J Biochem 183: 303-310).     

Kinetic characterization of the reconstituted tricarboxylate carrier from rat liver mitochondria

The tricarboxylate carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted in phospholipid vesicles by removing the detergent using hydrophobic chromatography on Amberlite. Optimal transport activity was obtained by using a Triton X-114/phospholipid ratio of 0.8, 6% cardiolipin and 24 passages through a single Amberlite column. In the reconstituted system the incorporated tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The activation energy of the exchange reaction was 70.1 kJ/mol. The rate of the exchange had a pH optimum between 7 and 8. The half-saturation constant was 0.13 mM for citrate and 0.76 mM for malate. All these properties were similar to those described for the tricarboxylate transport system in intact mitochondria. In proteoliposomes the maximum exchange rate at 25 degrees C reached 2000 mumols/min per g protein. This value was independent of the type of substrate present at the external or internal space of the liposomes (citrate or malate).     

Characterization of the unidirectional transport of carnitine catalyzed by the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria was purified by chromatography on hydroxyapatite and celite and reconstituted in egg yolk phospholipid vesicles by adsorbing the detergent on polystyrene beads. In the reconstituted system, in addition to the carnitine/carnitine exchange, the purified protein catalyzed a uni-directional transport (uniport) of carnitine measured as uptake into unloaded proteoliposomes as well as efflux from prelabelled proteoliposomes. In both cases the reaction followed a first-order kinetics with a rate constant of 0.023-0.026 min-1. Besides carnitine, also acylcarnitines were transported in the uniport mode. N-Ethylmaleimide inhibited the uni-directional transport of carnitine completely. The uniport of carnitine is not influenced by the delta pH and the electric gradient across the membrane. The activation energy for uniport was 115 kJ/mol and the half-saturation constant on the external side of the proteoliposomes was 0.53 mM. The maximal rate of the uniport at 25 degrees C was 0.2 mumol/min per mg protein, i.e. about 10 times lower than that of the reconstituted carnitine transport in exchange mode.     

Identification and purification of the tricarboxylate carrier from rat liver mitochondria

The tricarboxylate carrier from rat liver mitochondria was solubilized with Triton X-100 and purified by chromatography on hydroxyapatite and celite. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent Mr of 30,000. When reconstituted into liposomes, the tricarboxylate transport protein catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange. We obtained a 1070-fold purification with respect to the mitochondrial extract, the recovery was 22% and the protein yield 0.02%. The properties of the reconstituted carrier, i.e., requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the tricarboxylate transport system as characterized in intact mitochondria.     

Kinetics of the reconstituted 2-oxoglutarate carrier from bovine heart mitochondria

The 2-oxoglutarate carrier from the inner membrane of bovine heart mitochondria was purified by chromatography on hydroxyapatite/celite and reconstituted with egg yolk phospholipid vesicles by the freeze-thaw-sonication technique. In the reconstituted system the incorporated 2-oxoglutarate carrier catalyzed a first-order reaction of 2-oxoglutarate/2-oxoglutarate exchange. The substrate affinity for 2-oxoglutarate was determined to be 65 +/- 18 microM (15 determinations) and the maximum exchange rate at 25 degrees C reaches 4000-22,000 mumol/min per g protein, in dependence of the particular reconstitution conditions. The activation energy of the exchange reaction is 54.3 kJ/mol. The transport is independent of pH in the range between 6 and 8. When the first fraction of the hydroxyapatite/celite column eluate was used for reconstitution, besides the 2-oxoglutarate/2-oxoglutarate exchange, a significant activity of unidirectional uptake was observed. This activity may be due to a population of the carrier protein which is in a different state.     

Identification and purification of the aspartate/glutamate carrier from bovine heart mitochondria.

The aspartate/glutamate carrier from bovine heart mitochondria was solubilized with dodecyl-octaoxyethylene ether (C12E8) and purified by chromatography on hydroxyapatite and celite. On SDS gel electrophoresis, the purified aspartate/glutamate carrier consisted of a single protein band with an apparent Mr of 31,500. When reconstituted into liposomes the aspartate/glutamate carrier protein catalyzed an N-ethylmaleimide-sensitive aspartate/aspartate exchange. It was purified 620-fold with a recovery of 17.2% and a protein yield of 0.03% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e. requirement for a counteranion, substrate specificity and inhibitor sensitivity, were similar to those of the aspartate/glutamate carrier as characterized in mitochondria.     

Kinetics of the reconstituted dicarboxylate carrier from rat liver mitochondria

The dicarboxylate carrier from rat liver mitochondria was purified by the Amberlite/hydroxyapatite procedure and reconstituted in egg yolk phospholipid vesicles by removing the detergent with Amberlite. The efficiency of reconstitution was optimized with respect to the ratio of detergent/phospholipid, the concentration of phospholipid and the number of Amberlite column passages. In the reconstituted system the incorporated dicarboxylate carrier catalyzed a first-order reaction of malate/phosphate exchange. V of the reconstituted malate/phosphate exchange was determined to be 6000 mumol/min per g protein at 25 degrees C. This value was independent of the type of substrate present at the external or internal space of the liposomes (malate, phosphate or malonate). The half-saturation constant was 0.49 mM for malate, 0.54 mM for malonate and 1.41 mM for phosphate. The activation energy of the exchange reaction was determined to be 95.8 kJ/mol. The transport was independent of the external pH in the range between pH 6 and 8.     

Kinetics of the reconstituted 2-oxoglutarate carrier from bovine heart mitochondria

The 2-oxoglutarate carrier from the inner membrane of bovine heart mitochondria was purified by chromatography on hydroxyapatite / celite and reconstituted with egg yolk phospholipid vesicles by the freeze-thaw-sonication technique. In the reconstituted system the incorporated 2-oxoglutarate carrier catalyzed a first-order reaction of 2-oxoglutarate / 2-oxoglutarate exchange. The substrate affinity for 2-oxoglutarate was determined to be 65 ± 18 μM (15 determinations) and the maximum exchange rate at 25°C reaches 4000–22000 μmol / min per g protein, in dependence of the particular reconstitution conditions. The activation energy of the exchange reaction is 54.3 kJ / mol. The transport is independent of pH in the range between 6 and 8. When the first fraction of the hydroxyapatite / celite column eluate was used for reconstitution, besides the 2-oxoglutarate / 2-oxoglutarate exchange, a significant activity of unidirectional uptake was observed. This activity may be due to a population of the carrier protein which is in a different state.     

Kinetics of the Reconstituted Tricarboxylate Carrier from Eel Liver Mitochondria

The tricarboxylate carrier from eel liver mitochondria was purified by chromatography on hydroxyapatite and Matrix Gel Blue B and reconstituted into liposomes by removal of the detergent with Amberlite. Optimal transport activity was obtained by using a phospholipid concentration of 11.5 mg/ml, a Triton X-114/phospholipid ratio of 0.9, and ten passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased by cardiolipin and phosphatidylethanolamine and decreased by phosphatidylinositol. The reconstituted tricarboxylate carrier catalyzed a first-order reaction of citrate/citrate or citrate/malate exchange. The maximum transport rate of external [¹⁴C]citrate was 9.0 mmol/min per g of tricarboxylate carrier protein at 25°C and this value was virtually independent of the type of substrate present in the external or internal space of the liposomes. The half-saturation constant (K _m) was 62 M for citrate and 541 M for malate. The activation energy of the citrate/citrate exchange reaction was 74 kJ/mol from 5 to 19°C and 31 kJ/mol from 19 to 35°C. The rate of the exchange had an external pH optimum of 8.     

Partial Purification and Reconstitution of the alpha-Ketoglutarate Carrier from Corn (Zea mays L.) Mitochondria

The α-ketoglutarate carrier from corn shoot mitochondria (Zea mays L., B 73) was solubilized in Triton X-114 and partially purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. On SDS-gel electrophoresis, the hydroxyapatite/celite eluate showed various protein bands between 12 and 70 kilodaltons. When reconstituted into liposomes, the α-ketoglutarate transport protein catalyzed a phthalonate-sensitive α-ketoglutarate/α-ketoglutarate exchange. The protein was purified 60-fold with a recovery of 88% with respect to the mitochondrial extract. The protein yield was 0.6%. The properties of the reconstituted carrier, i.e. requirement for a counter-anion, substrate specificity, and inhibitor sensitivity, were similar to those of the α-ketoglutarate transport system as characterized in plant and animal mitochondria.     

Identification and purification of the carnitine carrier from rat liver mitochondria

The carnitine carrier from rat liver mitochondria, solubilized in Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by specific elution from celite in the presence of cardiolipin. On SDS-gel electrophoresis, the purified celite fraction consisted of a single band with an apparent Mr of 32,500. When reconstituted into liposomes the carnitine transport protein catalyzed an N-ethylmaleimide-sensitive carnitine/carnitine exchange. It was purified 970-fold with a recovery of 43% and a protein yield of 0.04% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., requirement for a countersubstrate, substrate specificity and inhibitor sensitivity, were similar to those of the carnitine transport system as characterized in intact mitochondria.     

Kinetic characterization of the reconstituted carnitine carrier from rat liver mitochondria

The carnitine carrier was purified from rat liver mitochondria and reconstituted into liposomes by removing the detergent from mixed micelles by Amberlite. Optimal transport activity was obtained with 1 microgram/ml and 12.5 mg/ml of protein and phospholipid concentration, respectively, with a Triton X-100/phospholipid ratio of 1.8 and with 16 passages through the same Amberlite column. The activity of the carrier was influenced by the phospholipid composition of the liposomes, being increased in the presence of cardiolipin and decreased in the presence of phosphatidylinositol. In the reconstituted system the incorporated carnitine carrier catalyzed a carnitine/carnitine exchange which followed a first-order reaction. The maximum transport rate of external [3H]carnitine was 1.7 mmol/min per g protein at 25 degrees C and was independent of the type of countersubstrate. The half-saturation constant (Km) for carnitine was 0.51 mM. The affinity of the carrier for acylcarnitines was in the microM range and depended on the carbon chain length. The activation energy of the carnitine/carnitine exchange was 133 kJ/mol. The carrier function was independent of the pH in the range between 6 and 8 and was inhibited at pH below 6.     

Purification and characterization of the reconstitutively active tricarboxylate transporter from rat liver mitochondria

The tricarboxylate transporter has been purified in reconstitutively active form from rat liver mitochondria. The transporter was extracted from mitoplasts with Triton X-114 in the presence of cardiolipin and citrate and was then purified by sequential chromatography on hydroxylapatite, Matrex Gel Orange A, Matrex Gel Blue B, and Affi-Gel 501. Analysis of the purified material via sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated the presence of one main protein band with an apparent molecular mass of 32.5 kDa. Upon incorporation into phospholipid vesicles, the purified transporter catalyzed a 1,2,3-benzenetricarboxylate-sensitive citrate/citrate exchange with a specific transport activity of 3240 nmol/4 min/mg of protein. This value was enhanced 831-fold with respect to the starting material. Substrate competition studies indicated that the reconstituted transport could be substantially inhibited by isocitrate, malate, and phosphoenolpyruvate, but not by alpha-ketoglutarate, succinate, malonate, pyruvate, or inorganic phosphate. Moreover, in addition to 1,2,3-benzenetricarboxylate, the reconstituted exchange was sensitive to the anion transport inhibitor n-butylmalonate but was insensitive to phenylsuccinate, alpha-cyano-4-hydroxycinnamate, and carboxyatractyloside. Finally, studies with covalent modifying agents indicated the purified transporter was inhibited by sulfhydryl reagents and by diethyl pyrocarbonate, 2,3-butanedione, phenylglyoxal, and pyridoxal 5-phosphate. In conclusion, these studies describe the first procedure to yield a highly purified tricarboxylate transport protein that both displays a high specific transport activity and can be obtained in quantities that readily enable further structural as well as functional studies. Based on its substrate specificity and inhibitor sensitivity, the purified 32.5-kDa protein appears to represent the complete tricarboxylate transport system found in rat liver mitochondria. Finally, new information is presented concerning the effect of covalent modifying reagents on the function of this transporter.     

Purification and characterization of the reconstitutively active adenine nucleotide carrier from mitochondria of Jerusalem artichoke (Helianthus tuberosus L.) tubers

The adenine nucleotide carrier from Jerusalem artichoke (Helianthus Tuberosus L.) tubers mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxapatite and Matrex Gel Blue B in the presence of cardiolipin and asolectin. SDS gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 33 kDa. When reconstituted in liposomes, the adenine nucleotide carrier catalyzed a pyridoxal 5-phosphate-sensitive ATP/ATP exchange. It was purified 75-fold with a recovery of 15% and a protein yield of 0.18% with respect to the mitochondrial extract. Among the various substrates and inhibitors tested, the reconstituted protein transported only ATP, ADP, and GTP and was inhibited by bongkrekate, phenylisothiocyanate, pyridoxal 5-phosphate, mersalyl and p-hydroxymercuribenzoate (but not N-ethylmaleimide). Atractyloside and carboxyatractyloside (at concentrations normally inhibitory in animal and plant mitochondria) were without effect in Jerusalem artichoke tubers mitochondria. V _max of the reconstituted ATP/ATP exchange was determined to be 0.53 mol/min per mg protein at 25°C. The half-saturation constant K _m and the corresponding inhibition constant K _i were 20.4 M for ATP and 45 M for ADP. The activation energy of the ATP/ATP exchange was 28 KJ/mol between 5 and 30°C. The N-terminal amino acid partial sequence of the purified protein showed a partial homology with the ANT protein purified from mitochondria of maize shoots.     

Purification of reconstitutively active alpha-oxoglutarate carrier from pig heart mitochondria

The alpha-oxoglutarate carrier from pig heart mitochondria has been solubilized with Triton X-114 and purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. When applied to SDS gel electrophoresis, the purified protein consists of only a single protein band with an apparent Mr of 31.5 kDa. It corresponds to band 4 of the five protein bands previously identified in the hydroxyapatite pass-through of Triton X-114 solubilized heart mitochondria (Bisaccia, F. and Palmieri, F. (1984) Biochim. Biophys. Acta 766, 386-394). When reconstituted into liposomes the alpha-oxoglutarate transport protein catalyzes a phthalonate-sensitive alpha-oxoglutarate/alpha-oxoglutarate exchange. It is purified 250-fold with a recovery of 62% and a protein yield of 0.1% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., the requirements for a counteranion, the substrate specificity and the inhibitor sensitivity, are similar to those described for alpha-oxoglutarate transport in mitochondria.     

Phosphorylation of bovine adrenodoxin. Structural study and enzymatic activity

Adrenodoxin is an iron-sulfur protein which functions as a carrier of reducing equivalents in steroid hydroxylation reactions catalyzed by specific cytochromes P-450 in steroidogenic tissues such as adrenal cortex. Purified bovine adrenocortical adrenodoxin was shown to be selectively phosphorylated upon incubation with purified cAMP-dependent protein kinase, whereas other protein kinases were ineffective. The phosphorylation reaction was completed within 45 min at 30 degrees C and resulted in the optimal incorporation of 1 mol phosphate/mol adrenodoxin. Apoadrenodoxin, lacking the iron-sulfur cluster, was also phosphorylated under similar conditions. An apparent Km of 55 microM with a Vmax of 0.3 pmol 32P incorporated min-1 mg adrenodoxin-1 was calculated. Phosphorylation resulted in a striking change in several molecular properties of adrenodoxin, such as electrophoretic behavior and hydroxyapatite affinity, thus providing the possibility of clearly separating phosphorylated from unphosphorylated adrenodoxin. In addition, phosphoadrenodoxin became refractory to mild trypsin degradation, whereas this was not the case with apoadrenodoxin. The phosphorylated site of adrenodoxin was identified as a serine residue; study of peptide products resulting from CNBr and proteolytic cleavages of phosphoadrenodoxin suggested that Ser-88 was the target of the phosphorylation reaction. The influence of phosphorylation upon adrenodoxin activity was examined using cholesterol side-chain cleavage and 11 beta-hydroxylase (11 beta) systems, reconstituted from purified components. Phosphorylation of adrenodoxin resulted in an average twofold decrease in its Km values for the two specific cytochromes P-450 involved. This effect was paralleled by a positive relationship between the degree of adrenodoxin phosphorylation and its ability to support the overall activity of reconstituted side-chain cleavage and 11 beta-hydroxylase systems. Although it remains to be examined whether adrenodoxin is phosphorylated in the intact cell, the present observations suggest that it represents a potential target in the hormonal regulation of the adrenocortical differentiated functions, especially by stimulatory agents acting through a cyclic-AMP-dependent mechanism, such as adrenocorticotropin.     

The Phosphate Transporter from Pea Mitochondria (Isolation and Characterization in Proteolipid Vesicles)

The phosphate transporter from mitochondria will exchange matrix phosphate for cytosolic phosphate and facilitate either phosphate/proton symport or phosphate/hydroxyl ion antiport. The phosphate transported into the matrix by this carrier is either used for ATP synthesis or exchanges back out to the cytosol on the dicarboxylate transporter, permitting entry of malate and succinate into the matrix. The phosphate transporter was solubilized from etiolated pea (Pisum sativum L. cv Alaska) mitochondrial membranes with Triton X-114, purified approximately 500-fold by hydroxylapatite chromatography, and reconstituted into azolectin vesicles that were preloaded with 0.1 or 10 mM phosphate. Phosphate transport was measured as the exchange of preloaded phosphate for external [32P]phosphate. Phosphate/phosphate exchange occurred for over 40 min at room temperature with an apparent K0.5 of 1.6 mM and a maximum velocity of over 700 nmol (mg protein)-1 min-1. Diethyl pyrocarbonate was used as an inhibitor-stop reagent. Transport was inhibited by p-hydroxyphenylglyoxal, p-hydroxymercuribenzoate, pyridoxal 5-phosphate, and dansyl chloride but was insensitive to sulfate, nitrate, and N-ethylmaleimide, the standard inhibitor for the mammalian phosphate transporter. Phosphate/hydroxyl exchange was stimulated when the proton gradient was collapsed with carbonyl cyanide m-chlorophenylhydrazone, but phosphate/phosphate exchange was unaffected by the uncoupler.     

Functional reconstitution of the partially purified aspartate-glutamate carrier from mitochondria

The aspartate/glutamate carrier from beef heart mitochondria has been solubilized with detergent. The transport protein was partially purified by chromatography on hydroxyapatite in the presence of dodecyl octaoxyethylene ether and high concentrations of ammonium acetate. During purification, the aspartate/glutamate carrier was identified by functional reconstitution into egg yolk phospholipid liposomes. After hydroxyapatite chromatography the protein is 30 fold enriched in aspartate/glutamate transport activity but still contains ADP/ATP-carrier and phosphate carrier. The reconstituted activity is specific for exchange of L-aspartate and L-glutamate and is similar to intact mitochondria with respect to substrate affinity and inhibitor sensitivity.     

Purification of reconstitutively active α-oxoglutarate carrier from pig heart mitochondria

The α-oxoglutarate carrier from pig heart mitochondria has been solubilized with Triton X-114 and purified by chromatography on hydroxyapatite and celite in the presence of cardiolipin. When applied to SDS gel electrophoresis, the purified protein consists of only a single protein band with an apparent M_r of 31.5 kDa. It corresponds to band 4 of the five protein bands previously identified in the hydroxyapatite pass-through of Triton X-114 solubilized heart mitochondria (Bisaccia, F. and Palmieri, F. (1984) Biochim. Biophys. Acta 766, 386–394). When reconstituted into liposomes the α-oxoglutarate transport protein catalyzes a phthalonate-sensitive α-oxoglutarate / α-oxoglutarate exchange. It is purified 250-fold with a recovery of 62% and a protein yield of 0.1% with respect to the mitochondrial extract. The properties of the reconstituted carrier, i.e., the requirements for a counteranion, the substrate specificity and the inhibitor sensitivity, are similar to those described for α-oxoglutarate transport in mitochondria.     

Isolation and reconstitution of the n-butylmalonate-sensitive dicarboxylate transporter from rat liver mitochondria

The mitochondrial dicarboxylate carrier has been substantially purified from rat liver mitoplasts by extraction with Triton X-114 in the presence of cardiolipin followed by chromatography on hydroxylapatite. Upon incorporation of the hydroxylapatite eluate into phospholipid vesicles, an n-butylmalonate-sensitive malonate/malate exchange has been demonstrated. This exchange activity is enhanced 226-fold relative to the starting material (i.e. detergent-extracted mitoplasts). Silver-stained sodium dodecyl sulfate-polyacrylamide gradient gels verify the high purity of this fraction relative to the starting material. Nonetheless, the banding pattern indicates that several protein species are still present. As isolated, the dicarboxylate transporter is rather unstable but can be stabilized either by the addition of 10% ethylene glycol and subsequent storage at -20 degrees C or by incorporation into phospholipid vesicles in the presence of malate followed by freezing in liquid nitrogen. Such proteoliposomes catalyze a [14C]malonate uptake which is characterized by a first order rate constant of 1.02 min-1 and a t 1/2 of 41 s. This uptake can be inhibited by dicarboxylates (e.g. succinate, malate, unlabeled malonate) but not by either alpha-ketoglutarate or by tricarboxylates (e.g. citrate, threo-Ds-isocitrate). Furthermore, the reconstituted malonate transport is dependent on internal malate and can be inhibited by n-butylmalonate, mersalyl, p-chloromercuribenzoate, and Pi, but not by N-ethylmaleimide. It is concluded that this highly purified fraction contains a reconstitutively active dicarboxylate transporter which, based on its substrate specificity and inhibitor sensitivity, appears to be identical to the native dicarboxylate transport system found in intact rat liver mitochondria.