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Biomedical subjects

F Palmieri

Publications and source records attributed to F Palmieri.

At least 55 records · Page 3Linked to original sources
PubMed1999

The purified and reconstituted ornithine/citrulline carrier from rat liver mitochondria catalyses a second transport mode: ornithine+/H+ exchange.

The mechanism of unidirectional transport of ornithine (i.e. in the absence of a counter-metabolite) has been investigated in proteoliposomes reconstituted with the ornithine carrier purified from rat liver mitochondria. The efflux of [(3)H]ornithine from proteoliposomes was stimulated by the addition of H(+) (but not of other cations) to the incubation medium. On keeping the pH in the compartment containing ornithine constant at 8.0, the flux of ornithine into or out of the proteoliposomes increased on decreasing the pH in the opposite compartment from 8.0 to 6.0. Ornithine influx was also stimulated when a higher H(+) concentration was generated inside the vesicles relative to the outside by the K(+)/H(+) exchanger nigericin in the presence of an outwardly directed K(+) gradient. A valinomycin-induced electrogenic flux of K(+) did not affect ornithine transport in the absence of a counter-metabolite. Furthermore, changes in fluorescence of the pH indicator pyranine, included inside the proteoliposomes, showed that the flux of ornithine is accompanied by translocation of H(+) in the opposite direction. It is concluded that the mitochondrial ornithine carrier catalyses an electroneutral exchange of ornithine(+) for H(+), in addition to the well-known 1:1 exchange of metabolites. Lysine(+), but not citrulline, can also be exchanged for H(+) by the ornithine carrier. The ornithine(+)/H(+) transport mode of the exchanger is an essential step in the catabolism of excess arginine.

Animals↗
PubMed1999

The mitochondrial dicarboxylate carrier is essential for the growth of Saccharomyces cerevisiae on ethanol or acetate as the sole carbon source.

The dicarboxylate carrier (DIC) is an integral membrane protein that catalyses a dicarboxylate-phosphate exchange across the inner mitochondrial membrane. We generated a yeast mutant lacking the gene for the DIC. The deletion mutant failed to grow on acetate or ethanol as sole carbon source but was viable on glucose, galactose, pyruvate, lactate and glycerol. The growth on ethanol or acetate was largely restored by the addition of low concentrations of aspartate, glutamate, fumarate, citrate, oxoglutarate, oxaloacetate and glucose, but not of succinate, leucine and lysine. The expression of the DIC gene in wild-type yeast was repressed in media containing ethanol or acetate with or without glycerol. These results indicate that the primary function of DIC is to transport cytoplasmic dicarboxylates into the mitochondrial matrix rather than to direct carbon flux to gluconeogenesis by exporting malate from the mitochondria. The delta DIC mutant may serve as a convenient host for overexpression of DIC and for the demonstration of its correct targeting and assembly.

Acetates↗
PubMed1999

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

The citrate carrier from maize (Zea mays) shoot mitochondria was solubilized with Triton X-100 and purified by sequential chromatography on hydroxyapatite and hydroxyapatite/celite in the presence of cardiolipin. SDS-gel electrophoresis of the purified fraction showed a single polypeptide band with an apparent molecular mass of 31 kD. When reconstituted into liposomes, the citrate carrier catalyzed a pyridoxal 5'-P-sensitive citrate/citrate exchange. It was purified 224-fold with a recovery of 50% and a protein yield of 0.22% with respect to the mitochondrial extract. In the reconstituted system the purified citrate carrier catalyzed a first-order reaction of citrate/citrate (0.065 min-1) or citrate/malate exchange (0.075 min-1). Among the various substrates and inhibitors tested, the reconstituted protein transported citrate, cis-aconitate, isocitrate, L-malate, succinate, malonate, glutarate, alpha-ketoglutarate, oxaloacetate, and alpha-ketoadipate and was inhibited by pyridoxal 5'-P, phenylisothiocyanate, mersalyl, and p-hydroxymercuribenzoate (but not N-ethylmaleimide), 1,2, 3-benzentricarboxylate, benzylmalonate, and butylmalonate. The activation energy of the citrate/citrate exchange was 66.5 kJ/mol between 10 degrees C and 35 degrees C; the half-saturation constant (Km) for citrate was 0.65 +/- 0.05 mM and the maximal rate (Vmax) of the citrate/citrate exchange was 13.0 +/- 1.0 micromol min-1 mg-1 protein at 25 degrees C.

Biological Transport↗
PubMed1998

The structure and organization of the human carnitine/acylcarnitine translocase (CACT1) gene2.

The carnitine/acylcarnitine translocase (CACT) transports acylcarnitines into mitochondria in exchange for free carnitine and it is, therefore, essential for the fatty acid beta-oxidation pathway. We have determined the exon-intron structure of the human CACT gene, which is responsible for a genetic disorder of fatty acid oxidation called CACT deficiency. The gene spans about 16.5 kb and consists of nine exons with the translation start site in exon 1. All the splice acceptor and donor sites conform to the AG/GT rules. All the introns except one are located at the level of the sequences coding for the extramembranous loops of CACT. We have designed a series of intronic oligonucleotide primers for amplifying each of the CACT exons together with their flanking intronic sequences, in segments well suited to detect mutations that would affect splicing of mRNA as well as the coding sequence itself.

Carnitine Acyltransferases↗
PubMed1998

The sequence, bacterial expression, and functional reconstitution of the rat mitochondrial dicarboxylate transporter cloned via distant homologs in yeast and Caenorhabditis elegans.

The dicarboxylate carrier (DIC) belongs to a family of transport proteins found in the inner mitochondrial membranes. The biochemical properties of the mammalian protein have been characterized, but the protein is not abundant. It is difficult to purify and had not been sequenced. We have used the sequence of the distantly related yeast DIC to identify a related protein encoded in the genome of Caenorhabditis elegans. Then, related murine expressed sequence tags were identified with the worm sequence, and the murine sequence was used to isolate the cDNA for the rat homolog. The sequences of the worm and rat proteins have features characteristic of the family of mitochondrial transport proteins. Both proteins were expressed in bacteria and reconstituted into phospholipid vesicles where their transport characteristics closely resembled those of whole rat mitochondria and of the rat DIC reconstituted into vesicles. As expected from the role of the DIC in gluconeogenesis and ureogenesis, its transcripts were detected in rat liver and kidney, but unexpectedly, they were also detected in rat heart and brain tissues where the protein may fulfill other roles, possibly in supplying substrates to the Krebs cycle.

Amino Acid Sequence↗
PubMed1998

Bacterial overexpression, purification, and reconstitution of the carnitine/acylcarnitine carrier from rat liver mitochondria.

The carnitine/acylcarnitine carrier from rat liver mitochondria was overexpressed in Escherichia coli. The expressed protein, recovered as inclusion bodies, was solubilized with sarkosyl and purified by Sephadex G-200 and celite chromatography. A yield of 15 mg of purified transport protein per liter of cell culture was obtained. Upon reconstitution into liposomes, the purified carrier catalyzed a [3H]carnitine/carnitine exchange inhibited by maleimides, mercurials, and sulfobetaines. Carnitine esters of various lengths were also transported. The Km for carnitine uptake was 0.47 +/- 0.11 mM, the Vmax of the exchange was 0.78 +/- 0.24 mmol/min per gram of protein, and the Ki for octanoylcarnitine was 13.5 +/- 4.3 microM. The transport properties of the recombinant carrier were virtually identical to those of the native transporter. These studies represent the first overexpression of the functionally active mitochondrial carnitine/acylcarnitine carrier, thus enabling structure/function analysis of this protein by site-directed mutagenesis.

Animals↗
PubMed1998

Expression in Escherichia coli, functional characterization, and tissue distribution of isoforms A and B of the phosphate carrier from bovine mitochondria.

The two isoforms of the mammalian mitochondrial phosphate carrier (PiC), A and B, differing in the sequence near the N terminus, arise from alternative splicing of a primary transcript of the PiC gene (Dolce, V., Iacobazzi, V., Palmieri, F., and Walker, J. E. (1994) J. Biol. Chem. 269, 10451-10460). To date, the PiC isoforms A and B have not been studied at the protein level. To explore the tissue-distribution and the potential functional differences between the two isoforms, polyclonal site-directed antibodies specific for PiC-A and PiC-B were raised, and the two bovine isoforms were obtained by expression in Escherichia coli and reconstituted into phospholipid vesicles. Western blot analysis demonstrated that isoform A is present in high amounts in heart, skeletal muscle, and diaphragm mitochondria, whereas isoform B is present in the mitochondria of all tissues examined. Heart and liver bovine mitochondria contained 69 and 0 pmol of PiC-A/mg of protein, and 10 and 8 pmol of PiC-B/mg of protein, respectively. In the reconstituted system the pure recombinant isoforms A and B both catalyzed the two known modes of transport (Pi/Pi antiport and Pi/H+ symport) and exhibited similar properties of substrate specificity and inhibitor sensitivity. However, they strongly differed in their kinetic parameters. The transport affinities of isoform B for phosphate and arsenate were found to be 3-fold lower than those of isoform A. Furthermore, the maximum transport rate of isoform B is about 3-fold higher than that of isoform A. These results support the hypothesis that the sequence divergence between PiC-A and PiC-B may have functional significance in determining the affinity and the translocation rate of the substrate through the PiC molecule.

Amino Acid Sequence↗
PubMed1998

Identification and purification of the reconstitutively active glutamine carrier from rat kidney mitochondria.

The glutamine carrier from rat kidney mitochondria, solubilized in dodecyl octaoxyethylene ether (C12E8) and partly purified on hydroxyapatite, was identified and completely purified by Celite chromatography. On SDS/PAGE, the purified glutamine carrier consisted of a single protein band with an apparent molecular mass of 41.5 kDa. When reconstituted into liposomes, the glutamine carrier catalysed both the unidirectional flux of glutamine and the glutamine/glutamine countertransport, which were completely inhibitable by a mixture of pyridoxal 5'-phosphate and N-ethylmaleimide. The carrier protein was purified 474-fold with a recovery of 58% and a protein yield of 0.12% with respect to the mitochondrial extract. The glutamine carrier-mediated transport is quite specific for l-glutamine. l-Asparagine is the only other amino acid that is efficiently transported by the reconstituted carrier protein. d-Glutamine, l-glutamate and l-aspartate are very poor substrates. The transport activity was inhibited by several thiol-group and amino-group reagents.

Animals↗
PubMed1998

Targeting and assembly of the oxoglutarate carrier: general principles for biogenesis of carrier proteins of the mitochondrial inner membrane.

We have studied the targeting and assembly of the 2-oxoglutarate carrier (OGC), an integral inner-membrane protein of mitochondria. The precursor of OGC, synthesized without a cleavable presequence, is transported into mitochondria in an ATP- and membrane potential-dependent manner. Import of the mammalian OGC occurs efficiently into both mammalian and yeast mitochondria. Targeting of OGC reveals a clear dependence on the mitochondrial surface receptor Tom70 (the 70 kDa subunit of the translocase of the outer mitochondrial membrane), whereas a cleavable preprotein depends on Tom20 (the 20 kDa subunit), supporting a model of specificity differences of the receptors and the existence of distinct targeting pathways to mitochondria. The assembly of minute amounts of OGC imported in vitro to the dimeric form can be monitored by blue native electrophoresis of digitonin-lysed mitochondria. The assembly of mammalian OGC and fungal ADP/ATP carrier occurs with high efficiency in both mammalian and yeast mitochondria. These findings indicate a dynamic behaviour of the carrier dimers in the mitochondrial inner membrane and suggest a high conservation of the assembly reactions from mammals to fungi.

Animals↗
PubMed1998

Human mitochondrial transmembrane metabolite carriers: tissue distribution and its implication for mitochondrial disorders.

Mitochondrial transmembrane carrier deficiencies are a recently discovered group of disorders, belonging to the so-called mitochondriocytopathies. We examined the human tissue distribution of carriers which are involved in the process of oxidative phosphorylation (adenine nucleotide translocator, phosphate carrier, and voltage-dependent anion channel) and some mitochondrial substrate carriers (2-oxoglutarate carrier, carnitine-acylcarnitine carrier, and citrate carrier). The tissue distribution on mRNA level of mitochondrial transport proteins appears to be roughly in correlation with the dependence of these tissues on mitochondrial energy production capacity. In general the main mRNA expression of carriers involved in mitochondrial energy metabolism occurs in skeletal muscle and heart. Expression in liver and pancreas differs between carriers. Expression in brain, placenta, lung, and kidney is lower than in the other tissues. Western and Northern blotting experiments show a comparable HVDAC1 protein and mRNA distribution for the tested tissues. Patient's studies showed that cultured skin fibroblasts may not be a reliable alternative for skeletal muscle in screening for human mitochondrial carrier defects.

Adenine Nucleotides↗
PubMed1998

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 degrees 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 (Km) was 62 microM for citrate and 541 microM for malate. The activation energy of the citrate/citrate exchange reaction was 74 kJ/mol from 5 to 19 degrees C and 31 kJ/mol from 19 to 35 degrees C. The rate of the exchange had an external pH optimum of 8.

Anguilla↗
PubMed1998

Role of human immunodeficiency virus in primary pulmonary hypertension--case reports.

Previous cases of pulmonary hypertension (PH) in human immunodeficiency virus (HIV) infection have been reported in the literature. The role of HIV in PH is still debatable. The purpose of this report was to analyze whether HIV plays a direct or indirect role in PH pathogenesis. Between February and November 1997, 56 HIV-infected patients with cardiac symptoms and signs were studied by serial color Doppler echocardiography. In four patients (7.1%), PH not related to other well-known associated conditions, was disclosed. In spite of a low serum HIV RNA viral load and a high-efficacy antiretroviral therapy, including a protease inhibitor in two patients, PH developed and worsened. It could be hypothesized that in some patients with an individual immunogenetic predisposition, a high secretion of cytokines and endothelin-1 stimulated by an unidentified pathogen different from HIV could lead to PH. Antiretroviral therapy seems not to prevent or reduce right ventricle pressure gradient in PH.

Adult↗
PubMed1998

Pulmonary cryptosporidiosis in patients with acquired immunodeficiency syndrome.

Several reports have showed Cryptosporidium species as a cause of intractable diarrhea and malabsorption in patients with acquired immunodeficiency syndrome (HIV). A case of chronic diarrhea in a drug addict woman associated with a symptomatic interstitial pulmonary infection due to Cryptosporidium parvum is described. This unusual C. parvum spread into the bronchial tree is underlined and a survey of the literature is made.

Acquired Immunodeficiency Syndrome↗