Biomedical subjects

Alessandra Olianas

Publications and source records attributed to Alessandra Olianas.

8 recordsLinked to original sources

PubMed2006

Barbary sheep (Ammotragus lervia): the structure of the adult beta-globin gene and the functional properties of its hemoglobin.

The adult beta-globin gene of Ammotragus lervia (Barbary sheep or aoudad) has been sequenced completely, including 185 base pairs of 5' flanking region and 168 base pairs 3' to the stop codon, and compared with that of other caprines such as goat (Capra hircus), wild Corsico-Sardinian (Ovis aries musimon) and Cypriot (O. a. ophion) mouflons, and domestic sheep (Ovis aries). The gene was identified as being located on a triplicated four-gene set cluster containing the HBBA locus (A-haplotype) as is the case of goat, sheep of the Hb A type and Corsico-Sardinian mouflon. Phylogenetic analyses support the evidence that caprines share a common ancestor that probably carried the A-haplotype and that a more recent deletion of a gene set gave rise to the duplicated cluster containing the HBBB locus (B-haplotype) found in sheep of the Hb B type and Cypriot mouflon, which evolved independently. Data also suggests that the Ammotragus beta-globin gene is older than genes of the examined caprines and indicates it followed an independent evolution after separating from species having the same HBBA locus. Similarly, phylogenetic analyses of beta-globin chain sequences suggest a different evolution for globins coded by the HBBA locus with respect to the HBBB. Ammotragus beta-globin chain shows all the amino acids responsible for the low oxygen affinity of ruminant Hbs. Investigations on the oxygen transport properties indicate that the intrinsic oxygen affinity of aoudad Hb is higher than the Hb B of the domestic sheep and, at the same time, more similar to that of other A type Hbs, whereas in the presence of the Cl(-) effector the oxygen affinity is approximately the same as that of the other species.

Amino Acid Sequence↗

PubMed2006

The hemocyanin of the shamefaced crab Calappa granulata: structural-functional characterization.

Arthropod hemocyanins (Hcs) transport and store oxygen and are composed of six subunits, or multiples thereof depending on the species. Calappa granulata Hc is found as a mixture of dodecamers (95%) and hexamers (5%). Removal of calcium ions and alkaline pH induce an incomplete partially reversible dissociation of dodecameric Hc. Two-dimensional electrophoretic pattern of dissociated Hc indicated a large heterogeneity in Hc subunit: most differences are likely to be explained by post-translational modifications. Dodecameric Hc showed a large Bohr effect (Deltalog P50/DeltapH = -0.95) and a normal cooperativity (h50 values = 2.7 +/- 0.2) in the presence of 10 mM CaCl2. The hexameric molecule displayed lower Bohr effect and cooperativity than the dodecamer. Lactate effect on the oxygen affinity (Deltalog P50 = 0.55) and the increase of lactate concentrations in animals kept in emersion were related to the increased oxygen requirements that occur during hypoxia in vivo. Calcium affects oxygen affinity only at high concentrations: this Hc appeared to lack the calcium high-affinity binding sites found in other species. The effect of temperature on both oxygen affinity and cooperativity was measured in the absence and presence of 10 mM lactate, allowing calculation of the exothermic contribution of lactate binding (DeltaH = -25 kJ mol(-1)).

Animals↗

PubMed2005

Functional and structural characterization of the myoglobin from the polychaete Ophelia bicornis.

The myoglobin of the polychaete annelid Ophelia bicornis was isolated, purified to homogeneity and characterized. The primary structure, obtained from cDNA and protein sequencing, consists of 139 amino acid residues. The alignment with other globin sequences showed that O. bicornis myoglobin misses the pre-A helix and the first six residues of the A helix. The presence of a PheB10-GlnE7 haem distal residue pair is in agreement with the measured oxygen affinity (P50=0.85 mmHg; 1 mmHg=0.133 kPa) and the only slightly higher autoxidation rate constant (0.28 h(-1)) with respect to that of the sperm whale myoglobin mutant E7 His-->Gln (0.21 h(-1)) and to elephant myoglobin (0.1 h(-1)). Oxygen-binding co-operativity was found to be absent under all the examined experimental conditions. The resistance of O. bicornis myoglobin towards autoxidation seems to confirm the important role of part of the A helix in the stability of the globin. The higher pKa of the acid-alkaline ferric transition of O. bicornis with respect to Asian elephant myoglobin, as well as the higher absorbance ratio of its ferric form to the oxy form measured in the Soret region (gammamet/gammaoxy) with respect to that of the African elephant myoglobin, suggested a stronger interaction between the distal glutamine and the water molecule at the sixth co-ordinate position.

Allosteric Regulation↗

PubMed2005

Tyrosinase activity and hemocyanin in the hemolymph of the slipper lobster Scyllarides latus.

The respiratory protein hemocyanin is present in molluscans and in some species of arthropods, and its dioxygen binding site strongly resembles that of the monophenol-hydroxylating and catechol-quinonising enzyme tyrosinase. Moreover, some hemocyanins show a certain extent of tyrosinase activity, so a common ancestry between the two proteins has been suggested. However, in the case purified hemocyanin of Scyllarides latus any attempts to evoke tyrosinase activity failed. A distinct tyrosinase has been purified to homogeneity from the hemolymph, and kinetically characterised. The purified tyrosinase showed both monophenolase and diphenolase enzyme activity and therefore it could be well defined as a true tyrosinase. This finding suggests that in the case of the studied crustacean the evolutionary functional divergence between dioxygen transport and oxidation of phenolics has already reached its completeness.

Animals↗

PubMed2005

Two sites for GTP binding in cathodic haemoglobins from Anguilliformes.

Cathodic haemoglobins of four species of anguilliform fish were characterized from a functional point of view, with special regard to the interaction with their physiological effectors. A series of oxygen-binding experiments at increasing GTP concentrations was carried out in order to compare GTP-binding activities in the absence and presence of saturating amounts of chloride. The results indicated that the cathodic haemoglobin of three species (Anguilla anguilla, Conger conger and Muraena helena) do have two sites for GTP-binding. In the absence of chloride, the two sites cannot be discriminated, whereas in the presence of chloride, a competition between the two anions occurred for the second GTP-binding site. The cathodic haemoglobin of Gymnothorax unicolor, which showed lower GTP sensitivity than the other haemoglobins examined, displayed only one GTP-binding site. The presence of an additional phosphate-binding site is not exceptional, although the way haemoglobin interacts with the two organic phosphate molecules may differ among species. This property may provide an auxiliary means of haemoglobin modulation for species that inhabit environments where oxygen availability is highly variable and haemoglobin-oxygen affinity needs to be modulated to different extents in order to satisfy physiological oxygen requirements.

Animals↗

PubMed2004

Oxygen-binding modulation of hemocyanin from the slipper lobster Scyllarides latus.

The oxygen-binding properties of hexameric hemocyanin (Hc) from Scyllarides latus were investigated with respect to pH, temperature, and modulating effect exerted by calcium, lactate, and urate. The oxygen affinity decreased at higher temperature, was slightly affected by pH, and was insensitive to lactate. Nevertheless, urate markedly increased Hc-oxygen affinity and its temperature sensitivity, acting as the physiological major positive effector: four urate sites per hexamer with an overall affinity constant of 1 x 10(4) M(-1) were found and the exothermic contribution of their binding was found to be about 30 kJ mol(-1). Calcium ions largely influenced oxygen affinity: their effect, which has an opposite sign at low (0-1 mM) and high (0.1-1 M) concentration ranges, indicates the presence of two independent types of binding sites with high and low affinity, respectively; however, only the former ones seem to be operative in vivo because, at physiological calcium concentrations, they are already saturated and the oxygen affinity is reduced.

Animals↗

PubMed2003

Structural-functional characterization of the cathodic haemoglobin of the conger eel Conger conger: molecular modelling study of an additional phosphate-binding site.

The protein sequence data for the alpha- and beta-chains have been deposited in the SWISS-PROT and TrEMBL protein knowledgebase under the accession numbers P83479 and P83478 respectively. The Conger conger (conger eel) haemoglobin (Hb) system is made of three components, one of which, the so-called cathodic Hb, representing approx. 20% of the total pigment, has been purified and characterized from both a structural and functional point of view. Stripped Hb showed a reverse Bohr effect, high oxygen affinity and slightly low cooperativity in the absence of any effector. Addition of saturating GTP strongly influences the pH dependence of the oxygen affinity, since the reverse Bohr effect, observed under stripped conditions, is converted into a small normal Bohr effect. A further investigation of the GTP effect on oxygen affinity, carried out by fitting its titration curve, demonstrated the presence of two independent binding sites. Therefore, on the basis of the amino acid sequence of the alpha- and beta-chains, which have been determined, a computer modelling study has been performed. The data suggest that C. conger cathodic Hb may bind organic phosphates at two distinct binding sites located along the central cavity of the tetramer by hydrogen bonds and/or electrostatic interactions with amino acid residues of both chains, which have been identified. Among these residues, the two Lys-alpha(G6) (where the letter refers to the haemoglobin helix and the number to the amino acid position in the helix) appear to have a key role in the GTP movement from the external binding region to the internal central cavity of the tetrameric molecule.

Allosteric Regulation↗

PubMed2003

Whale (Balaenoptera physalus) haemoglobin: primary structure, functional characterisation and computer modelling studies.

The functional properties of haemoglobin from the Mediterranean whale Balaenoptera physalus have been studied as functions of heterotropic effector concentration and temperature. Particular attention has been given to the effect of carbon dioxide and lactate since the animal is specialised for prolonged dives often in cold water. The molecular basis of the functional behaviour and in particular of the weak interaction with 2,3-diphosphoglycerate is discussed in the light of the primary structure and of computer modelling. On these bases, it is suggested that the A2 (Pro-->Ala) substitution observed in the beta chains of whale haemoglobin may be responsible for the displacement of the A helix known to be a key structural feature in haemoglobins that display an altered interaction with 2,3-diphosphoglycerate as compared with human haemoglobin. The functional and structural results, discussed in the light of a previous study on the haemoglobin from the Arctic whale Balaenoptera acutorostrata, give further insights into the regulatory mechanisms of the interactive effects of temperature, carbon dioxide and lactate.

Amino Acid Sequence↗