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.
"In detail, alkylation of thiol groups with 4-vinylpyridine and deacylation of the a-chain N terminus were carried out as described previously (D'Avino & di Prisco, 1989; Tamburrini et al., 1992, 1996). Alkylated globins were purified by reverse-phase HPLC, on a C4 Vydac column (4.6 9 250 mm). "
[Show abstract][Hide abstract] ABSTRACT: This study addresses the primary structure, the oxygen-binding properties and the CO-rebinding kinetics of the haemoglobins of the Patagonian toothfish Dissostichus eleginoides. D. eleginoides belongs to the family Nototheniidae, the most diversified of the suborder Notothenioidei, mostly exhibiting an Antarctic distribution. Some of its features are typical of Antarctic species, some are not. For instance, D. eleginoides appears not to have functional antifreeze glycoproteins (consistent with its non-Antarctic distribution). In contrast, it has a major and a minor haemoglobin (similar to many Antarctic notothenioids), and their very low oxygen affinity does not follow the trend of other non-Antarctic notothenioids and appears typical of cold-adapted species. Moreover, the amino-acid sequence reveals high identity with the globins of Antarctic notothenioids, arguing in favour of a common origin within notothenioids, and indicates that the primary structure of the major and minor haemoglobins has undergone modifications only to a limited extent. The ligand-rebinding kinetics of the major haemoglobin of D. eleginoides indicate a strong stabilisation of the quaternary T state at lower pH values.
"Fish constitute more than 23,000 species and live in wide range of environments (Helfman et al. 1997) and fish hemoglobins have shown a huge variation in components and oxygen-binding mechanisms (Weber 1982, 1996). For example, Tamburrini et al. (1992) studied the Antarctic fish hemoglobin and found that Gymnodraco acuticeps has a single hemoglobin without the Bohr effect, indicating that the role of this "
[Show abstract][Hide abstract] ABSTRACT: Cathepsin S is a lysosomal cysteine endopeptidase of the papain family. Our preliminary results showed the up-regulation of cathepsin S (CTSS) transcript during the early stage of Edwardsiella ictaluri infection, leading us to speculate that CTSS may play a role in infection. In this report, we identified, sequenced and characterized the channel catfish CTSS cDNA. Total RNA from tissues was isolated and cDNA libraries were constructed by the rapid amplification cDNA end (RACE) method. The gene-specific primers in conjunction with the RACE primers were used to PCR amplify 5'- and 3'-ends of the CTSS transcript. The complete channel catfish CTSS cDNA comprised 1530 nucleotides including a 96-nucleotide 5'-untranslated region (UTR), a 990-nucleotide open reading frame and a 444-nucleotide 3'-UTR. The open reading frame appears to encode a protein of 329 amino-acid residues with calculated molecular mass of 36.7kDa and pI of 5.96. The degree of conservation of the channel catfish CTSS amino-acid sequence in comparison to other species ranged from 56.6 to 68.5%. These results provide important information for further exploring the roles of channel catfish CTSS in antigen processing.
"The hydrolysate is suspended in 0.1% TFA and clarified by centrifugation. Separation of tryptic peptides (Tamburrini et al., 1992) is carried out by HPLC on a m-Bondapack C 18 reverse-phase column equilibrated with 0.1% TFA in water (solvent A) and 0.08% TFA in 99.92% acetonitrile (solvent B). Elution is carried out at a flow rate of 1 ml/min, and the eluate is monitored by measuring the absorbance at 220 and 280 nm. "
[Show abstract][Hide abstract] ABSTRACT: Because hemoglobins (Hbs) of all animal species have the same heme group, differences in their properties, including oxygen affinity, electrophoretic mobility, and pH sensitivity, must result from the interaction of the prosthetic group with specific amino acid residues in the primary structure. For this reason, fish globins have been the object of extensive studies in the past few years, not only for their structural characteristics but also because they offer the possibility to investigate the evolutionary history of Hbs in marine and freshwater species living in a large variety of environmental conditions. For such a purpose, phylogenetic analysis of globin sequences can be combined with knowledge of the phylogenetic relationships between species. In addition, Type I functional-divergence analysis is aimed toward predicting the amino acid residues that are more likely responsible for biochemical diversification of different Hb families. These residues, mapped on the three-dimensional Hb structure, can provide insights into functional and structural divergence.
Methods in Enzymology 02/2008; 436:539-70. DOI:10.1016/S0076-6879(08)36030-3 · 2.09 Impact Factor
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