M. Tamburrini

The amino acid sequence and oxygen-binding properties of the single hemoglobin of the cold-adapted Antarctic teleost Gymnodraco acuticeps

The complete amino acid sequence of the single hemoglobin of the Antarctic teleost Gymnodraco acuticeps has been determined. The alpha chain contains 142 amino acid residues; an acetylated seryl residue is at the amino terminal. The beta chain contains 146 residues. A very high degree of sequence identity has been found with hemoglobins of other Antarctic fishes. Oxygen binding is not modulated by pH and allosteric effectors. The Bohr and Root effects are absent, although specific amino acid residues, considered responsible of most of these functions, are conserved in the sequence, thus posing new questions about the molecular basis of these mechanisms. The low heat of oxygenation may be interpreted as one of the mechanisms involved in the process of cold adaptation.

Influence of the natural ripening stage, cold storage, and ethylene treatment on the protein and IgE-binding profiles of green and gold kiwi fruit extracts.

Kiwi fruit is an important source of food allergens, the number and relevance of which are still the object of investigation. Following a comparative analysis of the protein profiles in SDS-PAGE and IgE immunoblotting, a significant influence of conditions such as the ripening stage and the extraction method on the composition of green and gold kiwi fruit extracts was observed. Furthermore, the experimental data indicate that, mostly in the green species, a ripe fruit may have a different concentration of total proteins and a different amount of single components when ripeness is reached by different means of postharvest handling, such as ethylene exposure with or without previous cold storage. In summary, this study emphasizes the level of complexity associated with the preparation of extracts when a known and defined concentration of proteins/allergens is requested.

Kiwellin, a modular protein from green and gold kiwi fruits: Evidence of in vivo and in vitro processing and IgE binding

Kiwellin, an allergenic protein formerly isolated from green kiwi fruit, has been identified as the most abundant component of the gold kiwi species. A protein named KiTH, showing a 20 kDa band on reducing SDS-PAGE and 100% identity with the C-terminal region of kiwellin, has been identified in the extract of the ripe green species. In vitro treatment of purified kiwellin with the protease actinidin from green kiwi fruit originated KiTH and kissper, a recently described pore-forming peptide. Primary structure analysis and experimental evidence suggest that kiwellin is a modular protein with two domains. It may undergo in vivo proteolytic processing by actinidin, thus producing KiTH and kissper. When probed with sera recognizing kiwellin from green kiwi fruit, KiTH showed IgE binding, with reactivity levels sometimes different from those of kiwellin. The IgE-binding capacity of kiwellin from gold kiwi fruit appears to be similar to that of the green species.

The peculiar structural features of kiwi fruit pectin methylesterase: Amino acid sequence, oligosaccharides structure, and modeling of the interaction with its natural proteinaceous inhibitor

Pectin methylesterase (PME) from kiwi fruit (Actinidia deliciosa) is a glycoprotein, showing an apparent molecular mass of 50 kDa upon size exclusion chromatography and SDS‐PAGE. The primary structure, elucidated by direct sequencing of the protein, comprises 321 amino acid residues providing a molecular mass of 35 kDa. The protein has an acetylated Thr residue at the amino terminus and five N‐glycosylation consensus sequences, four of which are actually glycosylated. A careful investigation of the oligosaccharide structures demonstrated that PME glycans belong to complex type oligosaccharides essentially consisting of xylosylated polyfucosylated biantennary structures. Alignment with known mature plant PME sequences indicates that the postulated active site residues are conserved. Kiwi PME activity is inhibited following the interaction with the proteinaceous inhibitor PMEI, isolated from the same source. Gel‐filtration experiments show that kiwi PME/PMEI complex is stable in a large pH range and dissociates only at pH 10.0. Modeling of the interaction with the inhibitor was performed by using the crystal structure of the complex between kiwi PMEI and tomato PME as a template. The model shows that the binding site is the same reported for tomato PME. However, additional salt link interactions are found to connect the external loops of kiwi PME to PMEI. This finding may explain the higher pH stability of the complex formed by the two kiwi proteins respect to that formed by PMEI and tomato PME. Proteins 2008.

The Hemoglobins of the Antarctic Fishes Artedidraco orianae and Pogonophryne scotti AMINO ACID SEQUENCE, LACK OF COOPERATIVITY, AND LIGAND BINDING PROPERTIES

The oxygen-transport system of two species of Antarctic fishes belonging to the family Artedidraconidae,Artedidraco orianae and Pogonophryne scotti, was thoroughly investigated. The complete amino acid sequence of the α and β chains of the single hemoglobins of the two species was established. The oxygen-binding properties were also investigated, and were found not to differ significantly from those shown by blood, intact erythrocytes, and unstripped hemolysates. Both hemoglobins have unusually high oxygen affinity and display a relatively small Bohr effect; the Root effect is elicited only by organophosphates and is also reduced. Remarkably, the Hill coefficient is close to one in the whole pH range, indicating absence of cooperative oxygen binding which, in A. orianae hemoglobin, could be ascribed to the subunit heterogeneity shown upon oxygen dissociation. In comparison with the other families of the suborder Notothenioidei, the oxygen-transport system of these two species of Artedidraconidae has unique characteristics, which raise interesting questions on the mode of function of a multisubunit molecule and the relationship with cold adaptation.

Structure and function of hemoglobins from antarctic organisms: the search for correlations with adaptive evolution

For the study of temperature adaptations, Antarctica, more than any other habitat on earth, is indeed a unique natural laboratory. After 15 years of extensive investigations on the molecular basis of cold adaptation in antarctic marine and terrestrial organisms, we are finally able to begin identifying firm guidelines for understanding the interplay among biochemical/ physiological processes of oxygen transport, ecology and adaptive evolution.

Oxygen-Transport System and Mode of Life in Antarctic Fish

The separation of Antarctica from South America occurred 22–25 million years ago with the opening of the Drake Passage, and produced the Circum-Antarctic Current and the development of the Antarctic Polar Front. With the reduction of heat exchange from northern latitudes, cooling of the environment proceeded to the present extreme conditions. To date Antarctica is indeed a unique natural laboratory for the study of temperature adaptations and for understanding the interplay among biochemical/physiological processes, ecology and adaptive evolution.

Molecular Modelling Analysis of the Haemoglobins of the Antarctic Bird Catharacta maccormicki: the Hypothesis of a Second Phosphate Binding Site

Antarctic organisms are exposed to very low temperatures. Thus, in order to face extreme life conditions, suitable mechanisms of cold adaptation have been developed, involving physiological and biochemical processes [[1]].

Oxygen Transport in Diving Vertebrates

Oxygen transport proteins have developed, during evolution, complex regulatory molecular mechanisms to optimize the oxygenation-deoxygenation cycle according to the physiological needs of a given species. Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. In general, these mechanisms appear to be based on a thermodynamic linkage between binding of allosteric effectors (H+, CO2 and C1−, as well as organic and inorganic phosphates) and the basic reaction of hemoglobin with O2 [[1]]. For example, hemoglobins from Arctic mammals are characterized by a very low temperature sensitivity of oxygen binding, which has been interpreted as being beneficial to animals living in cold environments. Thus, with external temperature as low as −40°C, it is vital to reduce the overall AH of oxygen binding (generally exothermic, ΔH <0) as much as possible: deoxygenation will require much less heat and oxygen can still be released from the blood to the colder peripheral tissues. For this reason the hemoglobin of diving animals could be an interesting molecule for study since these mammals are specialized for prolonged dives, often in cold environments. We compared the hemoglobin systems from two species of whale: Balaenoptera physalus and Balaenoptera acutorostrata, which live in the Mediterranean and the Arctic seas, respectively. These species may have developed specific mechanisms for the maintenance of adequate oxygen supply to cold peripheral tissues in hypoxic conditions and in relation to their habitat.