Search PubMed⌕ Search

Biomedical subjects

F Bisaccia

Publications and source records attributed to F Bisaccia.

At least 19 recordsLinked to original sources
PubMed2003

Citrate uniport by the mitochondrial tricarboxylate carrier: a basis for a new hypothesis for the transport mechanism.

The tricarboxylate transport system located in the inner mitochondrial membrane was studied as an isolated protein reconstituted in proteoliposomes. The effects on the transport of citrate by various reagents, specific for different aminoacid residues, were analyzed. In the group of SH reagents, it was found that N-ethylmaleimide is an irreversible inhibitor of the citrate-citrate exchange, while HgCl2 and the mercurial mersalyl cause a rapid unidirectional efflux of citrate from liposomes. It was demonstrated that NEM and mercurials act on different SH groups. Dithioerythritol is not able to reverse the effect of mersalyl unless another reagent, pyridoxalphosphate, is present. Pyridoxalphosphate itself, a reagent specific for NH2 residues, is an effective inhibitor of citrate exchange transport, as measured in both influx and efflux, but it has no effect on the mercurial-induced efflux. The same behavior was observed with diethylpyrocarbonate, a reagent specific for histidine and tyrosine residues. Interestingly, a slow basic efflux of internal citrate, in the absence of countersubstrate, was observed in proteoliposomes. Because it is inhibited by the same reagents acting on the exchange process, it is deduced that it is catalyzed by the tricarboxylate carrier. The ability of the carrier to perform a uniport of the substrate suggests the presence of a single substrate binding site on the carrier protein. A preliminary kinetic approach indicates that such a transport model is compatible with this theory.

Animals↗
PubMed2002

Identification and reconstitution of the yeast mitochondrial transporter for thiamine pyrophosphate.

The genome of Saccharomyces cerevisiae contains 35 members of a family of transport proteins that, with a single exception, are found in the inner membranes of mitochondria. The transport functions of the 15 biochemically identified mitochondrial carriers are concerned with shuttling substrates, biosynthetic intermediates and cofactors across the inner membrane. Here the identification of the mitochondrial carrier for the essential cofactor thiamine pyrophosphate (ThPP) is described. The protein has been overexpressed in bacteria, reconstituted into phospholipid vesicles and identified by its transport properties. In confirmation of its identity, cells lacking the gene for this carrier had reduced levels of ThPP in their mitochondria, and decreased activity of acetolactate synthase, a ThPP-requiring enzyme found in the organellar matrix. They also required thiamine for growth on fermentative carbon sources.

Carbon↗
PubMed2002

Novel properties of peptides derived from the sequence coded by exon 26A of human elastin.

The exon 26A is a rarely expressed human elastin exon that codes for a hydrophilic and charged amino acid sequence. The functional role of elastin containing this additional sequence is unknown. The present investigation was aimed to determine the effect of synthetic peptides derived from this exon on the vascular tone of rat thoracic aorta. On phenilephrine-preconstricted rat thoracic aortic rings the peptides LSPELREGD and REGD cause dose-dependent relaxation in the concentration range from 10(-9) to 10(-5) M. omega-nitro-L-arginine methyl ester, a known inhibitor of the NO synthase, highly inhibits, although to a different extent, the relaxation induced by these peptides. Removal of endothelium and blocking of ATP-sensitive potassium channels by glibenclamide significantly inhibited the vasorelaxant activity of LSPELREGD but not that of REGD, suggesting a different mechanism of action and possibly a different receptor.

4-Aminopyridine↗
PubMed2000

Solution structure of the amino acid sequence coded by the rarely expressed exon 26A of human elastin: the N-terminal region.

We previously reported the structural and biological properties of the C-terminal sequence (REGDPSSSQHLPSTPSSPRV) coded by the rarely expressed exon 26A of human elastin. It assumes a stable type II beta-turn structure spanning the REGD sequence and possesses chemotactic and immunological properties. Here the structural characterization of the sequence coded by this exon was completed. Nuclear magnetic resonance and circular dichroism studies on the N-terminal amino acid sequence (GADEGVRRSLSPELREGD) showed the presence of an alpha-helix within VRRSL and a type II beta-turn within SPEL. The smaller peptides GADEGVRRSLSP and LSPELREGD revealed structural features similar to those identified in the parent peptide. No beta-turn was found in the REGD sequence of these peptides and no chemotactic activity was detected, thereby demonstrating that this biological activity is conformation dependent. Structural studies on additional peptides such as LREGD, ELREGD and LSPELREGDPSS showed that the presence of a Glu residue two positions before the Arg residue inhibits the beta-turn formation in the REGD sequence.

Amino Acid Sequence↗
PubMed1998

The amino acid sequence coded by the rarely expressed exon 26A of human elastin contains a stable beta-turn with chemotactic activity for monocytes.

The structural and biological properties of the amino acid sequence coded by the rarely expressed exon 26A of human elastin were investigated. The C-terminal portion of this sequence, corresponding to residues 600-619 of human tropoelastin, REGDPSSSQHLPSTPSSPRV and three shorter derived peptides, LREGDPSS, SSSQHLPS, and LPSTPSSP, were synthesized and studied. Spectroscopic analyses by CD and NMR have identified a type II beta-turn within the sequence REGD of the octapeptide LREGDPSS. This structural motif was found also in the tetrapeptide REGD in both trifluoroethanol and water. The CD spectrum of the tetrapeptide REGD in trifluoroethanol was consistent with a pure type II beta-turn. A high chemotactic activity for monocytes was exhibited by the structured peptides REGD (CI 0.90 at 10(-)7 M) and LREGDPSS (CI 0.80 at 10(-)11 M), at variance with the unfolded peptides LPSTPSSP and SSSQHLPS, suggesting that this activity is strictly correlated with folded structures. Because the exon 26A of human elastin is expressed in the neointima of hypertensive pulmonary arteries, and macrophages are present in this pathologic tissue [Liptay et al. (1993) J. Clin. Invest. 91, 588-594], the chemotactic activity for human monocytes reported in this paper is consistent with an active role played by the exon 26A in inducing the migration of the monocyte/macrophage cells to the neointima.

Amino Acid Sequence↗
PubMed1998

Identification of elastin peptides with vasorelaxant activity on rat thoracic aorta.

Elastin peptides obtained in vivo from the enzymatic degradation of elastic fibers are present in the circulating human blood. In order to verify the role that these peptides may have in the regulation of the vascular tone, the activity of several peptides identified in the elastolytic digest of human elastin and some of their structural homologues has been tested. Three of these peptides show a vasorelaxant activity in isolated rat aorta precontracted by phenylephrine. The activity observed is higher in the absence of the endothelium; in these conditions the IC50 for the peptides Val-Gly-Val-Ala-Pro-Gly, Val-Gly-Val-Pro-Gly and Val-Gly-Val-Hyp-Gly was 40 +/- 2, 73 +/- 2 and 10 +/- 1 ng/ml, respectively. They are active in the range of the pathological circulating concentration and their role could be important in the regulation of vascular tone during several elastin degradative diseases.

Animals↗
PubMed1997

Structure-activity relationships for some elastin-derived peptide chemoattractants.

In an attempt to explore the relationships between conformation of chemotactic peptides related to elastin and their biological activity we have studied five peptides: VGVAPG, VGVPG, VGAPG, GVAPG and GGVPG in solvents of different polarities which may mimic the environmental conditions at the receptor site. CD and NMR studies showed that GVAPG has no preference for structured conformations, while the other peptides may assume folded conformations in organic solvents. All these peptides but GGVPG showed chemotactic activity for monocytes. The chemotactic activity of VGVPG, VGAPG and VGVAPG was inhibited by lactose, while chemotaxis of peptide GVAPG was insensitive to lactose, suggesting the existence of different chemotactic receptors.

Chemotactic Factors↗
PubMed1996

Different recognition by clostripain of myelin basic protein in the lipid-free and lipid-bound forms.

Different proteolytic enzymes were tested for their ability to degrade the myelin basic protein of the central nervous system, purified in two different forms, the lipid-free form and the lipid-bound form. As shown by SDS gel electrophoresis only clostripain, a thiol protease, was able to distinguish between the two MBPs since it degraded MBP only in the lipid-free form. The failure to degrade lipid-bound MBP by clostripain could not be ascribed to the presence of lipids, since the other proteolytic enzymes tested degraded both MBPs independently from lipids giving fragments with different size. These results may be related to different conformations of MBPs possibly relevant for the study of myelin structure and antigenic properties of the protein.

Animals↗
PubMed1996

The mitochondrial oxoglutarate carrier protein contains a disulfide bridge between intramembranous cysteines 221 and 224.

The oxoglutarate carrier (OGC) purified from bovine heart mitochondria was treated, both in its active and in its SDS-denatured state, with the fluorescent N-(1-pyrenyl)maleimide and other SH reagents before and after reduction with dithioerythritol or beta-mercaptoethanol. The number of SH groups per OGC polypeptide chain was found to be about 1 for the oxidized carrier and 3 for the reduced carrier. The bovine oxoglutarate carrier contains three cysteines: Cys-184, Cys-221 and Cys-224. Sequencing of BrCN cleavage products of oxoglutarate carrier showed that N-(1-pyrenyl)maleimide binds to only Cys-184 of the oxidized protein and also to Cys-221 and Cys-224 after reduction of the protein. These results show the presence of a disulfide bridge between the latter two cysteines of the purified carrier. The oxidized and the reduced forms of the oxoglutarate carrier exhibited different Vmax but virtually the same K(m) values for oxoglutarate.

Amino Acid Sequence↗
PubMed1996

The mitochondrial oxoglutarate carrier: sulfhydryl reagents bind to cysteine-184, and this interaction is enhanced by substrate binding.

The interaction of sulfhydryl reagents with the oxoglutarate carrier (OGC) of bovine heart mitochondria was investigated in proteoliposomes reconstituted from purified carrier and lipids. Incubation of the proteoliposomes with maleimides or mercurials led to inhibition of the oxoglutarate carrier protein. The inhibition of oxoglutarate transport by mercurials was removed by dithioerythritol (DTE), whereas inhibition by maleimides was not. Preincubation of the proteoliposomes with mercurials protected the carrier protein against inactivation by the fluorescent sulfhydryl reagent N-(1-pyrenyl)maleimide (PM) and decreased the fluorescence associated with the carrier, indicating that mercurials bind to the same cysteine which is modified by PM. The presence of the substrates oxoglutarate and malate increased the binding of PM to the reconstituted carrier as well as the degree of inhibition of the reconstituted transport activity caused by PM, other maleimides, and mercurials. This result is consistent with the assumption that substrate binding causes a change in the tertiary structure of the carrier protein. The primary sequence of the oxoglutarate carrier contains three cysteines (Cys-184, Cys-221, and Cys-224). We provide evidence that PM labels only Cys-184, whereas Cys-221 and Cys-224 are linked by a disulfide bridge.

Amino Acid Sequence↗
PubMed1996

The formation of a disulfide cross-link between the two subunits demonstrates the dimeric structure of the mitochondrial oxoglutarate carrier.

Isolated oxoglutarate carrier (OGC) can be cross-linked to dimers by disulfide-forming reagents such as Cu2+-phenanthroline and diamide. Acetone and other solvents increase the extent of Cu2+ -phenanthroline-induced cross-linking of OGC. Cross-linked OGC re-incorporated in proteoliposomes fully retains the oxoglutarate transport activity. The amount of cross-linked OGC calculated by densitometry of scanned gels depends on the method of staining, since cross-linked OGC exhibits a higher sensitivity to Coomassie brilliant blue as compared to silver nitrate. Under optimal conditions the formation of cross-linked OGC dimer (stained with Coomassie brilliant blue) amounts to 75% of the total protein. Approximately the same cross-linking efficiency was evaluated from Western blots. Cross-linking of OGC is prevented by SH reagents and reversed by SH-reducing reagents, which shows that it is mediated by disulfide bridge(s). The formation of S-S bridge(s) requires the native state of the protein, since it is suppressed by SDS and by heating. Furthermore, the extent of cross-linking is independent of OGC concentration indicating that disulfide bridge(s) must be formed between the two subunits of native dimers. The number and localization of disulfide bridge(s) in the cross-linked OGC were examined by peptide fragmentation and subsequent cleavage of disulfide bond(s) by beta-mercaptoethanol. Our experimental results show that cross-linking of OGC is accomplished by a single disulfide bond between the cysteines 184 of the two subunits and suggest that these residues in the putative transmembrane helix four are fairly close to the twofold axis of the native dimer structure.

1-Propanol↗
PubMed1996

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).

Adenosine Diphosphate↗
PubMed1995

The N- and C-termini of the tricarboxylate carrier are exposed to the cytoplasmic side of the inner mitochondrial membrane.

Polyclonal antibodies were raised in rabbits against two synthetic peptides corresponding to the N- and C-terminal regions of the rat-liver mitochondrial tricarboxylate carrier. ELISA tests performed with intact and permeabilized rat-liver mitoplasts showed that both anti-N-terminal and anti-C-terminal antibodies bind only to the cytoplasmic surface of the inner membrane, indicating that both termini of the membrane-bound tricarboxylate carrier are exposed to the mitochondrial intermembrane space. Furthermore, tryptic digestion of intact mitoplasts markedly decreased the binding of anti-N-terminal and anti-C-terminal antibodies to the tricarboxylate carrier. These results are consistent with an arrangement of the tricarboxylate carrier monomer into an even number of transmembrane segments, with the N- and C-termini protruding toward the cytosol.

Amino Acid Sequence↗
PubMed1994

Transmembrane topography of the mitochondrial oxoglutarate carrier assessed by peptide-specific antibodies and enzymatic cleavage.

The folding of the peptide chain of the bovine heart oxoglutarate carrier in the inner mitochondrial membrane and in the membrane of reconstituted proteoliposomes has been investigated by enzymatic and immunochemical approaches using proteinase K and polyclonal site-directed antibodies, respectively. Two peptides corresponding to the amino acid sequences 2-12 (N-terminal peptide) and 303-314 (C-terminal peptide) have been synthesized and coupled to ovalbumin before being used to immunize rabbits. The specificity of the generated antibodies was tested by enzyme-linked immunosorbent assay (ELISA) and by Western blot analysis. Both anti-N-terminal and anti-C-terminal antibodies reacted specifically with the corresponding peptides and with the isolated oxoglutarate carrier, whereas only anti-C-terminal antibodies immunodetected the carrier in mitochondrial lysates and reacted with the membrane-bound carrier in mitoplasts and in freeze-thawed mitochondria. This result indicated that the last 12 C-terminal amino acid residues of the oxoglutarate carrier protein are accessible from the cytosolic side of the inner mitochondrial membrane. Anti-C-terminal antibodies did not recognize the oxoglutarate carrier in reconstituted proteoliposomes unless the membrane was inverted, indicating that the carrier was inserted unidirectionally in proteoliposomes, with an orientation opposite that found in mitochondria. The immunological data were complemented by data from a limited proteolysis study performed on the membrane-bound oxoglutarate carrier in proteoliposomes, using proteinase K. Cleavage of the carrier caused a time-dependent inhibition of the oxoglutarate-oxoglutarate exchange activity of the reconstituted system. Four cleavage sites were identified, between Val-39 and Gln-40, between Tyr-61 and Lys-62, between Phe-169 and Arg-170, and between Arg-182 and Gly-183.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Migration of monocytes in the presence of elastolytic fragments of elastin and in synthetic derivates. Structure-activity relationships.

YGVG and GLVPG, two new chemokinetic peptides, were identified in elastolytic digests of elastin, besides the known chemoattractant hexapeptide VGVAPG. In order to identify possible sequences responsible for the chemotactic and chemokinetic activities and to obtain structure-activity relationships we synthesized some analogues of these peptides: FGVG (an analogue of YGVG), GVAPG and VGAPG (derived from the hexapeptide by deletion of Val1 or Val3). FGVG has a higher chemotactic activity than YGVG (chemotactic indices of 0.62 and 0.49, respectively, at 10(-11) M) and is both chemotactic and chemokinetic. Checkerboard analysis demonstrated that both peptides derived from the hexapeptide present, in addition to the chemotactic activity, a chemokinetic activity. The chemotactic index of GVAPG is 0.66 at 10(-10) M, while for VGAPG it is 0.86 at 10(-9) M. These results indicate that the deletion of the N-terminal residue of the elastin chemotactic peptides, VGVAPG and GFGVG, gives rise to chemokinetic activity. CD and NMR studies showed that all peptides are largely unordered in aqueous solution.

Amino Acid Sequence↗
PubMed1993

Reaction mechanism of the reconstituted tricarboxylate carrier from rat liver mitochondria.

Transport of citrate and malate by the tricarboxylate carrier from rat liver mitochondria has been studied in a reconstituted system. Homologous citrate/citrate antiport and heterologous (electroneutral) citrate/malate antiport was kinetically analyzed. The maximal rates of the two exchange modes did not vary significantly within pH 7.0 to 7.8 which is the optimum pH-range for transport activity. On the other hand, the apparent transport affinity varied considerably within this range. Calculations on the basis of the different pK values for citrate and malate indicate that only H-citrate2- and malate2- are accepted as transport species by the tricarboxylate carrier. A complete set of half-saturation constants was established for citrate and malate on both the external and the internal side of the membrane. Both the Km and Vmax for citrate and malate were independent of the nature of the countersubstrate at the other side of the membrane. Bisubstrate initial velocity analyses of the exchange reaction resulted in a kinetic pattern which is consistent with a sequential antiport mechanism. This type of mechanism implies formation of a ternary complex of the carrier with two substrate molecules before the transport reaction occurs. Thus the tricarboxylate carrier falls into the functional family of mitochondrial carrier proteins showing sequential transport mechanisms.

Animals↗