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F Palmieri

Publications and source records attributed to F Palmieri.

At least 19 recordsLinked to original sources
PubMed1992

The mitochondrial carnitine carrier: characterization of SH-groups relevant for its transport function.

The transport function of the purified and reconstituted carnitine carrier from rat liver mitochondria was correlated to modification of its SH-groups by various reagents. The exchange activity and the unidirectional transport, both catalyzed by the carnitine carrier, were effectively inhibited by N-ethylmaleimide and submicromolar concentrations of mercurial reagents, e.g., mersalyl and p-(chloromercuri)benzenesulfonate. When 1 microM HgCl2 or higher concentrations of the above mentioned mercurials were added, another transport mode of the carrier was induced. After this treatment, the reconstituted carnitine carrier catalyzed unidirectional substrate-efflux and -influx with significantly reduced substrate specificity. Control experiments in liposomes without carrier or with inactivated carrier protein proved the dependence of this transport activity on the presence of active carnitine carrier. The mercurial-induced uniport correlated with inhibition of the 'physiological' functions of the carrier, i.e., exchange and substrate specific unidirectional transport. The effect of consecutive additions of various reagents including N-ethylmaleimide, mercurials, Cu(2+)-phenanthroline and diamide on the transport function revealed the presence of at least two different classes of SH-groups. N-Ethylmaleimide blocked the carrier activity by binding to SH-groups of one of these classes. At least one of these SH-groups could be oxidized by the reagents forming S-S bridges. Besides binding to the class of SH-groups to which N-ethylmaleimide binds, mercurials also reacted with SH-groups of the other class. Modification of the latter led to the induction of the efflux-type of carrier activity characterized by loss of substrate specificity.

Animals↗
PubMed1992

Identification and purification of the ornithine/citrulline carrier from rat liver mitochondria.

The ornithine/citrulline carrier from rat liver mitochondria, solubilized with Triton X-100 and partially purified on hydroxyapatite, was identified and completely purified by PD-10, DEAE-Sephacel and celite chromatography. On SDS/polyacrylamide gel electrophoresis, the purified ornithine/citrulline carrier consisted of a single protein band with an apparent molecular mass of 33.5 kDa. When reconstituted into liposomes the ornithine carrier protein catalyzed an active mersalyl sensitive ornithine/ornithine exchange. It was purified 438-fold with a recovery of 38% and a protein yield of 0.09% with respect to the extract derived from mitoplasts. The purified and reconstituted protein did not catalyze a significant unidirectional transport of ornithine. Citrulline was found to be the best countersubstrate for the transport of ornithine, followed by lysine and arginine. The exchange activity was inhibited by several sulphydryl reagents.

Animals↗
PubMed1992

The cleavable presequence is not essential for import and assembly of the phosphate carrier of mammalian mitochondria but enhances the specificity and efficiency of import.

The phosphate carrier (PiC) of mammalian mitochondria is synthesized with a cleavable presequence, in contrast to other members of the mitochondrial family of inner membrane carrier proteins. The precursor of PiC is efficiently imported, proteolytically processed, and correctly assembled in isolated mitochondria. Here we report that a presequence-deficient PiC was imported with an efficiency of about 50% as compared with the authentic precursor of PiC. This mature-sized PiC was correctly assembled, demonstrating that the presequence is not essential for the assembly pathway. We found the following functions for the PiC presequence. (i) The presequence by itself was able to target a passenger protein to mitochondria with a low efficiency, suggesting that the mammalian PiC contains multiple targeting signals, the more efficient one(s) present in the mature protein part. (ii) Deletion of the presequence allowed a more efficient heterologous import of mammalian PiC into mitochondria from Saccharomyces cerevisiae and Neurospora crassa, indicating an important role of the presequence in determining the specificity of PiC import. (iii) Import of the presequence-deficient PiC required a higher membrane potential across the inner membrane than that of the presequence-carrying form. Therefore, the presequence also enhances the translocation of PiC into the inner membrane.

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↗
PubMed1992

Transmembrane arrangement of mitochondrial porin or voltage-dependent anion channel (VDAC).

Porin or voltage-dependent anion-selective channel (VDAC) is the main protein responsible for the high permeability of the outer mitochondrial membrane. The mitochondrial porin is mainly composed of sided beta-strands, in analogy with bacterial porin, whose structure has been resolved at 1.8 A resolution. In mitochondrial porins the N-terminal region forms an amphipathic alpha-helix, a structure conserved in organisms very distant from the evolutionary point of view. This part of the protein is exposed to the water phase, as demonstrated by ELISA assays. Various extramembranous loops have been identified by specific proteolytic cleavages. These overall, combined results were used to draw a model of the transmembrane arrangement of mammalian porin. This model is compared to other mitochondrial and bacterial porin models.

Amino Acid Sequence↗
PubMed1992

Sequence and pattern of expression of a bovine homologue of a human mitochondrial transport protein associated with Grave's disease.

A human cDNA has been isolated previously from a thyroid library with the aid of serum from a patient with Grave's disease. It encodes a protein belonging to the mitochondrial metabolite carrier family, referred to as the Grave's disease carrier protein (GDC). Using primers based on this sequence, overlapping cDNAs encoding the bovine homologue of the GDC have been isolated from total bovine heart poly(A)+ cDNA. The bovine protein is 18 amino acids shorter than the published human sequence, but if a frame shift requiring the removal of one nucleotide is introduced into the human cDNA sequence, the human and bovine proteins become identical in their C-terminal regions, and 308 out of 330 amino acids are conserved over their entire sequences. The bovine cDNA has been used to investigate the expression of the GDC in various bovine tissues. In the tissues that were examined, the GDC is most strongly expressed in the thyroid, but substantial amounts of its mRNA were also detected in liver, lung and kidney, and lesser amounts in heart and skeletal muscle.

Amino Acid Sequence↗
PubMed1992

Sequences of the human and bovine genes for the mitochondrial 2-oxoglutarate carrier.

The oxoglutarate carrier transports 2-oxoglutarate across the inner membranes of mitochondria in an electroneutral exchange for malate or other dicarboxylic acids. The sequences of its human and bovine genes have been determined from overlapping genomic clones generated by polymerase chain reactions by use of primers and probes based on the bovine cDNA sequence. The bovine and human genes are split into 6 and 8 exons, respectively, and five introns are found in the same positions in both genes. The coding and protein sequences are 93% and 96.6% identical, respectively. The human oxoglutarate carrier protein is 314 amino acids in length and, in common with the bovine protein, does not appear to have a processed presequence to help to target it into mitochondria.

Amino Acid Sequence↗
PubMed1991

Characterization of SH groups in porin of bovine heart mitochondria. Porin cysteines are localized in the channel walls.

Porin from bovine heart mitochondria contains probably two cysteines (Cys126 and Cys230 in human porin, Kayser, H., Kratzin, H. D., Thinnes, F. P., Götz, H., Schmidt, W. E., Eckart, K. & Hilschmann, N. (1989) Biol. Chem. Hoppe-Seyler 370, 1265-1278). Reduced and oxidized forms of these cysteines were investigated in purified protein and in intact mitochondria using the agents dithioerythritol, cuprous(II) phenantroline, diamide and performic acid. Furthermore, intact mitochondria were labelled with the sulfhydryl-alkylating agents N-[14C]ethylmaleimide, eosin-5-maleimide and N-(1-pyrenyl)-maleimide. Affinity chromatography of bovine heart porin was performed with cysteine-specific material. The results can be summarized as follows: (1) Porin has one reduced and two oxidized forms of apparent molecular masses between 30 and 35 kDa. The native form of porin is the reduced 33 kDa form. The oxidized forms only appear after denaturation with SDS. (2) The 35-kDa reduced and the 33.5-kDa oxidized forms of porin show the same pore-forming properties after reconstitution of the protein into lipid bilayer membranes. (3) Labelling of cysteines by eosin-5-maleimide and N-(1-pyrenyl)-maleimide suggested their location at a boundary between the water-phase and the lipid-phase. Incubation of intact mitochondria with N-ethylmaleimide prior to eosin-5-maleimide and N-(1-pyrenyl)maleimide treatment resulted in the inhibition of the fluorescent labelling. Among the cysteines present in the primary structure, Cys126 is the most sensitive to N-ethylmaleimide binding. (4) Bovine heart mitochondrial porin covalently bound to Affi-Gel 501 (with a 1.75 nm long spacer), but not to Thiopropyl-Sepharose 6B (with a 0.51 nm spacer). This suggests that at least one of the cysteines is localized between 0.51 nm and 1.75 nm deep in the protein micelle.

Animals↗
PubMed1991

Peptide-specific antibodies and proteases as probes of the transmembrane topology of the bovine heart mitochondrial porin.

We have investigated the transmembrane topology of the bovine heart mitochondrial porin by means of proteases and antibodies raised against the amino-terminal region of the protein. The antisera against the human N-terminus reacted with porin in Western blots of NaDodSO4-solubilized bovine heart mitochondria and with the membrane-bound porin in enzyme-linked immunosorbent assay (ELISA). The immunoreaction with mitochondria coated on microtiter wells showed that the amino-terminal region of the protein is not embedded in the lipid bilayer but is exposed to the cytosol. Back-titration of unreacted anti-N-terminal antibodies after their incubation with intact mitochondria demonstrated that the porin N-terminus is also exposed in "noncoated" mitochondria. No difference in antisera reactivity was observed between intact and broken mitochondria. Intact and broken mitochondria were subjected to proteolysis by specific proteases. The membrane-bound bovine heart porin was strongly resistant to proteolysis, but a few specific cleavage sites were observed. Staphylococcus aureus V8 protease gave a large 24K N-terminal peptide, trypsin produced a 12K N-terminal and an 18K C-terminal peptide, and chymotrypsin gave two peptides of Mr 19.5K and 12.5K, which were both recognized by the antiserum against the human N-terminus. Carboxypeptidase A was ineffective in cleaving the membrane-bound porin in both intact and broken mitochondria. Thus, the carboxy-terminal part of the protein is probably not exposed to the water phase. The cleavage patterns of membrane-bound porin, obtained with S. aureus V8 protease, trypsin, and chymotrypsin, showed no difference between intact and broken mitochondria, thus indicating that all porin molecules have the same orientation in the membrane. The computer analysis of the sequence of human B-lymphocyte porin suggested that 16 beta-strands can span the phospholipid bilayer. This result, together with the overall information presented, allowed us to draw a possible scheme of the transmembrane arrangement of mammalian mitochondrial porin.

Amino Acid Sequence↗
PubMed1991

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.

Animals↗
PubMed1991

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.

Animals↗
PubMed1991

Reaction mechanism of the reconstituted oxoglutarate carrier from bovine heart mitochondria.

The transport mechanism of the reconstituted oxoglutarate carrier, purified from bovine heart mitochondria, was studied kinetically. A complete set of half-saturation constants (Km) was established for the two different substrates oxoglutarate and malate on both the external and the internal sides of the membrane. The internal affinities for oxoglutarate (Km 0.17 mM) and malate (Km 0.7 mM) were higher than the corresponding external affinities (Km 0.3 mM and 1.4 mM, respectively). The exclusive presence of a single transport affinity for each substrate on one side of the membrane indicated a unidirectional insertion of the oxoglutarate carrier into the liposomal membrane. The Km values and also the maximum exchange rates (8-11 mumol.min-1.mg protein-1) for oxoglutarate and malate were independent of the nature of the counter substrate on the other side of the membrane. Under these defined conditions we analyzed the antiport mechanism in two-reactant initial velocity studies varying both the internal and external substrate concentrations. From the kinetic patterns obtained, a sequential type of mechanism became evident, implying that one internal and one external substrate molecule form a ternary complex with the carrier before transport occurs. A quantitative analysis of substrate interaction with the unloaded or single-substrate-occupied carrier revealed that rapid-equilibrium random conditions were fulfilled, characterized by a fast and independent binding of internal and external substrate. This kinetic mechanism agrees with previous results obtained in intact mitochondria. Considering also the data available for other mitochondrial carriers, a common kinetic mechanism (sequential type) for this carrier family is suggested.

Animals↗
PubMed1991

Biogenesis of the mitochondrial phosphate carrier.

The mitochondrial phosphate carrier (PiC) is a member of the family of inner-membrane carrier proteins which are generally synthesized without a cleavable presequence. Surprisingly, the cDNA sequences of bovine and rat PiC suggested the existence of an amino-terminal extension sequence in the precursor of PiC. By expressing PiC in vitro, we found that PiC is indeed synthesized as a larger precursor. This precursor was imported and proteolytically processed by mitochondria, whereby the correct amino-terminus of the mature protein was generated. Import of PiC showed the characteristics of mitochondrial protein uptake, such as dependence on ATP and a membrane potential and involvement of contact sites between mitochondrial outer and inner membranes. The precursor imported in vitro was correctly assembled into the functional form, demonstrating that the authentic import and assembly pathway of PiC was reconstituted when starting with the presequence-carrying precursor. These results are discussed in connection with the recently postulated role of PiC as an import receptor located in the outer membrane.

Amino Acid Sequence↗
PubMed1991

Transmembrane topography of the mitochondrial phosphate carrier explored by peptide-specific antibodies and enzymatic digestion.

Two peptides corresponding to the amino acid sequences 1-10 (N-terminal peptide) and 303-313 (C-terminal peptide) of the bovine heart mitochondrial phosphate carrier have been synthesized. After being coupled to ovalbumin, they were injected into rabbits to raise polyclonal antibodies. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and/or Western blot. Anti-N-terminal antibodies and anti-C-terminal antibodies exclusively reacted with the corresponding terminal peptide, they also reacted with the isolated phosphate carrier as well as with the phosphate carrier protein in mitochondrial lysates. Both anti-N-terminal and anti-C-terminal antibodies bound to freeze-thawed mitochondria, indicating that both termini of the membrane-bound phosphate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane. These immunological data were complemented with results concerning enzymatic cleavage of the membrane-bound phosphate carrier by carboxypeptidase A and by an arginine-specific endoprotease. Carboxypeptidase A markedly decreased the binding of anti-C-terminal antibodies to phosphate carrier in freeze-thawed mitochondria. Arg-endoprotease cleaved the phosphate carrier in inside-out submitochondrial particles, but not in right-side-out particles, yielding two fragments of similar apparent molecular weight (Mr approximately equal to 14.5K), which were immunodetected only by the anti-N-terminal antiserum, and a fragment of Mr approximately equal to 17K which was detected only by the anti-C-terminal antiserum. It appears, therefore, that Arg-endoprotease cleavage sites of the phosphate carrier are present only at the matrix side of the inner mitochondrial membrane, at Arg-140 and/or Arg-152.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Characterization of pore-forming activity in liver mitochondria from Anguilla anguilla. Two porins in mitochondria?

A fast purification procedure for the isolation and purification of eukaryotic porin (De Pinto et al., (1987) Biochim. Biophys. Acta 905, 499-502) was applied to liver mitochondria of the fish Anguilla anguilla. A protein preparation was obtained which formed slightly anionically selective pores in reconstitution experiments with lipid bilayer membranes. The distribution of single-channel conductances had two maxima of 2.4 nS and 4.0 nS in 1 M KCl. Sodium dodecylsulfate electrophoretograms of the protein preparation showed the presence of two bands of very similar electrophoretic mobility (32 and 32.5 kDa). Both bands cross-reacted with antibodies raised against purified bovine heart porin and with antibodies raised against the 19 amino acids N-terminal end of human porin. No cross-reactivity was observed with antibodies against yeast porin. The peptide maps of the two bands showed slight differences. The possibility of the presence of two different porins in liver mitochondria of Anguilla anguilla is discussed. An extensive immunological comparison of different mitochondrial porins is presented.

Anguilla↗
PubMed1991

Nucleotide sequence of a human heart cDNA encoding the mitochondrial phosphate carrier.

We have isolated and characterized a full length cDNA clone encoding the precursor of the human heart mitochondrial phosphate carrier protein. The entire clone is 1330 bp in length with 5'- and 3'-untranslated regions of 48 and 184 bp, respectively. The open reading frame encodes the mature protein consisting of 312 amino acids, preceded by a presequence of 49 amino acids. The amino acid sequence of the mature human phosphate carrier is 93.6, 94.2 and 33.6% identical to that of the phosphate carrier from beef, rat and yeast, respectively. Like other mitochondrial transport proteins, the human phosphate carrier has a tripartite structure. Each of the three repeats contains two hydrophobic regions which presumably span the membrane in the form of alpha-helices.

Amino Acid Sequence↗
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↗