Search PubMed⌕ Search

Biomedical subjects

F Bisaccia

Publications and source records attributed to F Bisaccia.

32 records · Page 2Linked to original sources
PubMed1993

Transmembrane topology, genes, and biogenesis of the mitochondrial phosphate and oxoglutarate carriers.

Phosphate and oxoglutarate carriers transport phosphate and oxoglutarate across the inner membranes of mitochondria in exchange for OH- and malate, respectively. Both carriers belong to the mitochondrial carrier protein family, characterized by a tripartite structure made up of related sequences about 100 amino acids in length. The results obtained on the topology of the phosphate and oxoglutarate carriers are consistent with the six alpha-helix model proposed by Saraste and Walker. In both carriers the N- and C-terminal regions are exposed toward the cytosol. In addition, the oxoglutarate carrier has been shown to be a dimer by means of crosslinking studies. The bovine and human genes coding for the oxoglutarate carrier are split into eight and six exons, respectively, and five introns are found to the same position in both genes. The bovine and human phosphate carrier genes have the same organization with nine exons separated by eight introns at exactly the same positions. The phosphate carrier of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to the oxoglutarate carrier and the other members of the mitochondrial carrier family. The precursor of the phosphate carrier is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. The presequence-deficient phosphate carrier is imported with an efficiency of about 50% as compared with the precursor of the phosphate carrier and is correctly assembled, demonstrating that the mature portion of the phosphate carrier contains sufficient information for import and assembly into mitochondria.

Amino Acid Sequence↗
PubMed1993

Functional properties of purified and reconstituted mitochondrial metabolite carriers.

Eight mitochondrial carrier proteins were solubilized and purified in the authors' laboratories using variations of a general procedure based on hydroxyapatite and Celite chromatography. The molecular mass of all the carriers ranges between 28 and 34 kDa on SDS-PAGE. The purified carrier proteins were reconstituted into liposomes mainly by using a method of detergent removal by hydrophobic chromatography on polystyrene beads. The various carriers were identified in the reconstituted state by their kinetic properties . A complete set of basic kinetic data including substrate specificity, affinity, interaction with inhibitors, and activation energy was obtained. These data closely resemble those of intact mitochondria, as far as they are available from the intact organelle. Mainly on the basis of kinetic data, the asymmetric orientation of most of the reconstituted carrier proteins were established. Several of their functional properties are significantly affected by the type of phospholipids used for reconstitution. All carriers which have been investigated in proteoliposomes function according to a simultaneous (sequential) mechanism of transport; i.e., a ternary complex, made up of two substrates and the carrier protein, is involved in the catalytic cycle. The only exception was the carnitine carrier, where a ping-pong mechanism of transport was found. By reaction of particular cysteine residues with mercurial reagents, several carriers could be reversibly converted to a functional state different from the various physiological transport modes. This "unphysiological" transport mode is characterized by a combination of channel-type and carrier-type properties.

Animals↗
PubMed1992

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.

Amino Acid Transport Systems, Acidic↗
PubMed1990

Sequence of the bovine 2-oxoglutarate/malate carrier protein: structural relationship to other mitochondrial transport proteins.

The amino acid sequence of the 2-oxoglutarate/malate carrier protein, a component of the inner membranes of mitochondria, has been deduced from the sequences of overlapping cDNA clones. These clones were generated in polymerase chain reactions using, in the first instance, complex mixtures of oligonucleotides as primers and probes, with sequences based upon partial protein sequences of cyanogen bromide fragments of the purified protein. The protein sequence of the carrier, including the initiator methionine, is 314 amino acids long. The mature protein has a modified alpha-amino group, but the nature of this modification and the precise position of the mature N-terminal amino acid have not been ascertained, although it must lie in amino acids 1-4 of the deduced protein sequence. Comparison of the protein sequence with itself and with those of 3 other mitochondrial carrier proteins, ADP/ATP translocase, the phosphate carrier, and the uncoupling protein from brown fat, shows that all 4 proteins contain a 3-fold repeated sequence about 100 amino acids in length, and all the repeats are interrelated. This suggests that the members of this family of proteins have similar structures and mechanisms and that they have evolved from a common origin. The distribution of hydrophobic amino acids in the oxoglutarate/malate carrier supports the view that the domains are folded into similar structural motifs, possibly consisting of two transmembrane alpha-helices joined by an extensive extramembranous hydrophilic region. Clones of cDNA arising from a longer related transcript of the oxoglutarate/malate carrier gene have also been analyzed. They contain 271 additional nucleotides in the 3' noncoding region.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

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).

Animals↗
PubMed1989

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.

Animals↗
PubMed1988

Purification and reconstitution of two anion carriers from rat liver mitochondria: the dicarboxylate and the 2-oxoglutarate carrier.

Two anion-transporting systems, i.e., the dicarboxylate carrier and the 2-oxoglutarate carrier, have been purified from rat liver mitochondria and functionally identified. The dicarboxylate carrier has been isolated in active form by hydroxyapatite chromatography after partial removal of the solubilizing detergent Triton X-114 from the mitochondrial extract. The SDS gel electrophoresis of this preparation consists mainly of one protein band with an apparent Mr of 28,000, identified as the dicarboxylate carrier. Complete purification of the 28 kDa protein in inactive form has been achieved by sequential chromatography on hydroxyapatite and Celite followed by SDS extraction of the retained protein. The 2-oxoglutarate carrier has been purified by hydroxyapatite chromatography after extensive removal of Triton X-114 from the detergent extract. SDS gel electrophoresis of the purified fraction shows a single band with an apparent Mr of 32,500. When reconstituted into liposomes, the functional properties of the two isolated carrier proteins resemble closely those of the dicarboxylate and the 2-oxoglutarate transport systems characterized in mitochondria.

Animals↗
PubMed1987

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.

Animals↗
PubMed1985

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.

Animals↗
PubMed1984

Specific elution from hydroxylapatite of the mitochondrial phosphate carrier by cardiolipin.

The role of cardiolipin in the purification of the mitochondrial phosphate carrier by hydroxylapatite has been investigated. Without added cardiolipin, the reconstituted phosphate-transport activity in the hydroxylapatite eluate is small and only confined to the first fraction. With cardiolipin added to the extract, the eluted activity is much higher and present until fraction 6. The activity retained by hydroxylapatite in the absence of cardiolipin is eluted after addition of this phospholipid to the column. The requirement of added cardiolipin diminishes on increasing the concentration of solubilized mitochondria. The hydroxylapatite eluate contains five protein bands in the Mr-region of 30 000-35 000, which are differently distributed in the various fractions. Among these, only the presence and the relative amount of band 3 of Mr 33 000 corresponds to the phosphate transport activity. Cardiolipin is the only phospholipid tested which causes elution of band 3 from hydroxylapatite; on the other hand, it prevents the elution of band 2 and retards that of band 5 (the ADP/ATP carrier). Band 1 starts to appear in the second fraction even without cardiolipin. On increasing the concentration of cardiolipin, in the first fraction of the hydroxylapatite eluate band 3 increases and the contamination of band 4 decreases. Under optimal conditions a preparation of band 3 about 90% pure and with high reconstituted phosphate transport activity is obtained. It is concluded that the elution of the phosphate carrier from hydroxylapatite requires cardiolipin and that the phosphate carrier is identical with (or with part of) band 3 of the hydroxylapatite eluate.

Animals↗