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Aim: A cytosolic carbonic anhydrase (I-CA) isoform, showing higher catalytic rate with respect to temperate counterparts, was isolated from the gills of the haemoglobinless, circulating CA deprived Antarctic teleost Chionodraco hamatus. To understand the molecular basis of the high I-CA activity, a structural characterisation has been undertaken. Methods: I-CA was purified from gill epithelium by sulphanilamide sepharose gel FPLC. The amino acid sequence of I-CA was established by direct protein sequencing. Isoelectric focusing (IEF) and Western blot analysis were used to show post-translational modifications of I-CA. Thermodynamic parameters of CA were calculated by measuring carbonic anhydrase activity in the 0-40 °C range by a radioactive assay. Sequence analysis and computational procedures were performed. Results: I-CA consists of 259 residues and its molecular mass deduced from the amino acid sequence (28449 Da) was higher than the value obtained by mass spectrometry (2873829225 Da). Treatment with dithiothreitol (DTT) abolished this discrepancy, indicating possible post-translational modifications also confirmed by IEF analysis. S-glutathionylation of I-CA was demonstrated by using specific anti-GSH antibody. Deglutathionylated form of I-CA maintained its activity despite a higher susceptibility to H2O2. Thermodynamic parameters of I-CA showed some traits of cold-adaptation as described for other enzymes from Antarctic fish. The I-CA sequence also suggests specific adaptive features related to the environmental temperature. Conclusion: A novel enzyme variant with high turnover rate was found in the respiratory epithelium of the icefish. The evolution of this branchial isoform could correlate with cold adaptation and the absence of haemoglobin and circulating CA in this fish.

Ciliates comprise species synthesizing water-diffusible mating type factors or pheromones and species synthesizing insoluble, cell membrane-bound pheromones. Euplotes crassus has traditionally been placed in the latter group. In contrast with this notion, we found that E. crassus is a constitutive pheromone-secreting ciliate, like other Euplotes species. From cell-free filtrate preparations of the E. crassus strain L-2D, we isolated two distinct pheromones, designated as Ec-± and Ec-1, and determined their complete amino acid sequences by combined chemical and genetic approaches. The Ec-± pheromone sequence extends for 56 amino acid residues with six cysteines and shows a molecular mass of 6,183 Da, while the Ec-1 pheromone sequence extends for 45 amino acid residues with 10 cysteines and shows a molecular mass of 4,840 Da. Marked structural differences distinguish the full-length Ec-± and Ec-1 coding sequences, which have been cloned and characterized from the transcriptionally active macronuclear genome. They were taken as clear indication that the Ec-± and Ec-1 pheromones are specified by genes that are not allelic, but likely derived from a duplicated genetic locus of the transcriptionally silent micronuclear genome

"Background: Food allergy is increasingly common worldwide. Tools for allergy diagnosis measuring IgE improved much since allergenic molecules and microarrays started to be used. IgE response toward allergens belonging to the same group of molecules has not been comprehensively explored using such approach yet. Objective: Using the model of lipid transfer proteins (LTPs) from plants as allergens, including two new structures, we sought to define how heterogeneous is the behavior of homologous proteins. Methods: Two new allergenic LTPs, Act d 10 and Act c 10, have been identified in green (Actinidia deliciosa) and gold (Actinidia chinensis) kiwifruit (KF), respectively, using clinically characterized allergic patients, and their biochemical features comparatively evaluated by means of amino acid sequence alignments. Along with other five LTPs from peach, mulberry, hazelnut, peanut, mugwort, KF LTPs, preliminary tested positive for IgE, have been immobilized on a microarray, used for IgE testing 1,003 allergic subjects. Comparative analysis has been carried out. Results: Alignment of Act d 10 primary structure with the other allergenic LTPs shows amino acid identities to be in a narrow range between 40 and 55%, with a number of substitutions making the sequences quite different from each other. Although peach LTP dominates the IgE immune response in terms of prevalence, epitope recognition driven by sequence heterogeneity has been recorded to be distributed in a wide range of behaviors. KF LTPs IgE positive results were obtained in a patient subset IgE positive for the peach LTP. Anyhow, the negative results on homologous molecules allowed us to reintroduce KF in patients’ diet. Conclusion: The biochemical nature of allergenic molecule belonging to a group of homologous ones should not be taken as proof of immunological recognition as well. The availability of panels of homologous molecules to be tested using microarrays is valuable to address the therapeutic intervention."

Proteomic is the study of the overall protein content of a cell, a tissue or an organism, aiming to identification and qualification of all the expressed proteins. Many studies, mainly linked to the development of new medicines or therapies, have so far been focused on the analysis of human proteins. On the other hand, little attention has been given to plant tissues, and in particular to fruit ones. In this study, a proteomic approach was used for the analysis of biodiversity of fruits of some Mediterranean species, and in particular: apple, cv Annurca Rossa del Sud, cv Annurca tradizionale and cv Golden Delicious; peach, cv Percoco di Tunisi; lemon, cv Femminello Siracusano; persimmon, cv Calfuyu; orange, cv Washington Navel; grape, cv Moscato Bianco; prickly pear, cv Gialla. For each sample, the total protein concentration of the saline extract of the pulp was determined, and the protein electrophoretic pattern of the extract was analysed by SDS-PAGE. The fruits under investigation showed a variable protein content, from 20mg/100g of prickly pear to 0.54mg/100g of grape. For each species, a low number of abundant protein, having different molecular weights, was shown. In some cases, a significant variability, dependent on the post-harvesting time, was found. Proteins present in high amounts were analysed by N-terminal amino acid sequencing, and the obtained data were compared to sequences available in Data Banks. The results showed that most of the abundant proteins are involved in mechanisms of plant defence against water and temperature stress, pathogen attack, chemical agents, etc.

The hemolysate of the Antarctic teleost Gobionotothen gibberifrons (family Nototheniidae) contains two hemoglobins (Hb 1 and Hb 2). The concentration of Hb 2 (15-20% of the total hemoglobin content) is higher than that found in most cold-adapted Notothenioidei. Unlike the other Antarctic species so far examined having two hemoglobins, Hb 1 and Hb 2 do not have globin chains in common. Therefore this hemoglobin system is made of four globins (two alpha- and two beta-chains). The complete amino-acid sequence of the two hemoglobins (Hb 1, alpha2(1)beta2(1); Hb 2, alpha2(2)beta2(2)) has been established. The two hemoglobins have different functional properties. Hb 2 has lower oxygen affinity than Hb 1, and higher sensitivity to the modulatory effect of organophosphates. They also differ thermodynamically, as shown by the effects on the oxygen-binding properties brought about by temperature variations. The oxygen-transport system of G. gibberifrons, with two functionally distinct hemoglobins, suggests that the two components may have distinct physiological roles, in relation with life style and the environmental conditions which the fish may have to face. The unique features of the oxygen-transport system of this species are reflected in the phylogeny of the hemoglobin amino-acid sequences, which are intermediate between those of other fish of the family Nototheniidae and of species of the more advanced family Bathydraconidae.

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-á(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.

The Arctic fish Anarhichas minor, a benthic sedentary species, displays high emoglobin multiplicity. The three major hemoglobins (Hb 1, Hb 2, and Hb 3) show important functional differences in pH and organophosphate regulation, subunit cooperativity, and response of oxygen binding to temperature. Hb 1 and Hb 2 display a low, effector-enhanced Bohr effect and no Root effect. In contrast, Hb 3 displays pronounced Bohr and Root effects, accompanied by strong organophosphate regulation. Hb 1 has the beta (beta(1)) chain in common with Hb 2; Hb 3 and Hb 2 share the alpha (alpha(2)) chain. The amino acid sequences have been established. Several substitutions in crucial positions were observed, such as Cys in place of C-terminal His in the beta(1) chain of Hb 1 and Hb 2. In Hb 3, Val E11 of the beta(2) chain is replaced by Ile. Homology modeling revealed an unusual structure of the Hb 3 binding site of inositol hexakisphoshate. Phylogenetic analysis indicated that only Hb 2 displays higher overall similarity with the major Antarctic hemoglobins. The oxygen transport system of A. minor differs remarkably from those of Antarctic Notothenioidei, indicating distinct evolutionary pathways in the regulatory mechanisms of the fish respiratory system in the two polar environments.

Free-living species of ciliated Protozoa control their vegetative (mitotic) proliferation and mating (sexual) processes by diffusible, cell type-specific protein signals (pheromones). One of these molecules, designated En-2, was isolated from a species, Euplotes nobilii, living in the stably cold marine waters of Antarctica, and its complete amino acid sequence of 60 residues was determined by automated Edman degradation of the whole protein and peptides generated by trypsin digestion. The proposed sequence is: DIEDFYTSETCPYKNDSQLA(20)WDTCSGGTGNCGTVCCGQCF(40)SFPVSQSCAGMADSNDCPNA(60). The En-2 structure appears to be characterized by an adaptive insertion of a glycine-rich motif potentially capable to confer more flexibility to a functionally critical region of the molecule.

This research dealing with fish, bird and mammal hemoglobins is in the framework of the study of the molecular basis of cold adaptation in Antarctic organisms. The study of hemoglobin structural and functional properties in Antarctic teleosts allowed a correlation of amino acid sequence, multiplicity, and oxygen-binding features with ecological constraints. Amino acid sequences of alpha and beta chains were analysed to build phylogenetic trees, while the organisation and expression of globin genes was studied. The respiratory proteins of Antarctic birds were investigated, in relation with the oxygen demands arising from their characteristic behaviour (diving or flight) and, in general, from the extreme conditions of the Antarctic habitat. The study of Weddell seal hemoglobins indicated that the combined effect of carbon dioxide, organic phosphates and temperature optimises oxygen delivery to all tissues in spite of their relative heterothermia. The crystallographic structure of the carbonmonoxy derivative of Trematomus newnesi major hemoglobin was resolved, giving new insight into the study of the Root effect. The molecular models of skua hemoglobins revealed the presence of a second, additional phosphate binding site located between the two alpha chains, paving the way to further studies.

Pagothenia borchgrevinki, has a higher haemoglobin concentration than other Antarctic notothenioids and the high oxygen capacity may correlate with the relatively active mode of life of this fish. The fish has five haemoglobins (Hb C, Hb 0, Hb 1, Hb 2 and Hb 3) with Hb 1 accounting for 70–80% of the total, and Hb C being present in trace amounts. Hb 1 and Hb 2 are functionally similar in terms of Bohr and Root effects. Hb 3 has a weaker Bohr effect than Hb 1 and Hb 2, and the Root effect is similar to that of Hb 1. Hb 0 has a strong Bohr effect and the Root effect is enhanced to a larger extent by the physiological effectors chlorides and phosphates than that of the other components with the exception of Hb C. The heats of oxygenation are lower than those of temperate fish haemoglobins. Temperature variations may have a different effect on the functional properties of each haemoglobin, and chloride and phosphates may play an important role in the conformational change between the oxy and deoxy structures. The complete amino acid sequences of Hb 1 and Hb 0, as well as partial N-terminal or internal sequences of the other haemoglobins, have been established. The high multiplicity of functionally distinct haemoglobins indicates that P. borchgrevinki, has a specialized haemoglobin system.

In order to study the molecular mechanisms of enzyme cold adaptation, direct amino acid sequence, catalytic features, thermal stability and thermodynamics of the reaction and of heat inactivation of L-glutamate dehydrogenase (GDH) from the liver of the Antarctic fish Chaenocephalus aceratus (suborder Notothenioidei, family Channichthyidae) were investigated. The enzyme shows dual coenzyme specificity, is inhibited by GTP and the forward reaction is activated by ADP and ATP. The complete primary structure of C. aceratus GDH has been established; it is the first amino acid sequence of a fish GDH to be described. In comparison with homologous mesophilic enzymes, the amino acid substitutions suggest a less compact molecular structure with a reduced number of salt bridges. Functional characterisation indicates efficient compensation of Q10, achieved by increased kcat and modulation of S0:5, which produce a catalytic efficiency at low temperature very similar to that of bovine GDH at its physiological temperature. The structural and functional characteristics are indicative of a high extent of protein flexibility. This property seems to find correspondence in the heat inactivation of Antarctic and bovine enzymes, which are inactivated at very similar temperature, but with different thermodynamics.

Abstract: We purified an esterase from Bacillus acidocaldarius whose NH2-terminal sequence corresponds to an open reading frame (ORF) previously reported to show homology with the mammalian hormone sensitive lipase (HSL)-like subgroup of the esterase/lipase family. The enzyme displays esterase but not lipase activity. For this enzyme we predicted the w',8-hydrolase topological fold common to several esterases and lipases and identified Ser 155, Asp 252 and His 282 as the putative members of the catalytic triad.