Electrophoretic analysis of Antarctic fish from South Georgia

White muscle samples of Antarctic fish (Notothenia rossii, N. neglecta, N. gibberifrons and Chaenocephalus aceratus) were subjected to electrophoretic tests. Isoelectric focusing of general muscle proteins provides a quick method of identifying species. Six muscle enzymes were resolved by horizontal starch gel electrophoresis. These are presumed to be the products of ten independent gene loci. Coefficients of genetic distance were calculated and substantially agree with the conventional taxonomy. Levels of genetic variability are within the normal range for marine teleosts. Two polymorphic enzymes. GPI and PGM, might prove useful as 'genetic tags' for stock identification.     

Electrophoretic analysis of Antarctic fish from South Georgia

White muscle samples of Antarctic fish ( Notothenia rossii, N., neglecta. N. gibbe-rifrons and Chaenocephalus (aceratus ) were subjected to electrophoretic tests. lsoelectric focusing of general muscle proteins provides a quick method of iden-tyfying species. Six muscle enzymes were resolved by horizontal starch gel clectrophoresis. These are presumed to be the products of ten independent gene loci. Coefficients of genetic distance were calculated and substantially agree with the conventional taxnomy. Levels of genetic variability are within the normal range for marine teleosts. Two polymorphic enzymes. GPI and PGM. might prove useful as 'genetic tags' for stock identification.     

Functionally distinct haemoglobins of the cryopelagic Antarctic teleost Pagothenia borchgrevinki

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.     

Aspartic proteinases in Antarctic fish

The present review surveys several recent studies of the aspartic proteinases from Antarctic Notothenioidei, a dominating fish group that has developed a number of adjustments at the molecular level to maintain metabolic function at low temperatures. Given the unique peculiarities of the Antarctic environment, studying the features of Antarctic aspartic proteinases could provide new insights into the role of these proteins in fish physiology. We describe here: (1) the biochemical properties of a cathepsin D purified from the liver of the hemoglobinless icefish Chionodraco hamatus; (2) the biochemical characterization of Trematomus bernacchii pepsins variants A1 and A2 obtained by heterologous expression in bacteria; and (3) the identification of two closely related, novel aspartic proteinases from the liver of the two Antarctic fish species mentioned above. Overall, the results show that Notothenioidei aspartic proteinases display a number of characteristics that are remarkably different from those of mammalian aspartic proteinases, including high turnover number or high catalytic efficiency. We have named the newly identified aspartic proteinases "Nothepsins" and classified them relative to aspartic proteinases from other species.     

Freezing-point depressing peptides and glycoproteins from Arctic-boreal and Antarctic fish

Summary Antifreeze glycoproteins have been isolated from the Antarctic Nototheniid Trematomus hansoni and the Chaenichthyid species Chionodraco hamatus and Chaenocephalus aceratus. Their molecular weights range from 7,400–62,000 Daltons. The amino acid and sugar composition of these glycoproteins indicate that they consist of the same subunits which are known from related Nototheniids. In the Arctic-boreal sculpin Myoxocephalus scorpius two antifreeze peptides with a molecular weight of 6,000–7,000 Dalton could be isolated from the skin, which is likely to act as a barrier against initiation of ice propagation during contact with ice crystals in the water.     

Antifreeze glycoproteins from Antarctic fish. Inactivation by borate.

Antifreeze glycoprotein, which has previously been shown to be inactive in the presence of borate, migrates electrophoretically as the borate complex, presumably through formation of borate complexes with hydroxyl groups on the sugar side chains. Antifreeze glycoprotein (5 mg/ml) has been found to be completely active in the presence of 0.1 M borate at pH 7, but inactive at pH 9. A titration curve of pH versus the antifreeze activity of glycoprotein (5 mg/ml) in 0.1 M borate showed a progressive decrease in antifreeze activity as the pH was increased. Concomitant with decreases in activity were increases in binding of borate. At pH 9.0, nearly 2 mol of borate were complexed per glycotripeptide. Ultracentrifuge analyses showed similar molecular weights and laser quasi-elastic light scattering showed similar diffusions at pH 7.0 and 9.0 in borate and in the absence of borate. The binding of borate, rather than a change in conformation, is thus directly related to the loss of antifreeze activity. Alkaline borate also decreased hemagglutinating activity of Osage orange lectin and decreased the inhibition of the activity by the antifreeze glycoproteins.     

Mitochondria-rich cells in Antarctic fish gills

We used transmission and scanning electron microscopy and an antibody to the inner mitochondrial membrane to study the presence and localisation of mitochondria-rich cells in the gills of two Antarctic fishes, Chionodraco hamatus (Channichthyidae) and Trematomus bernacchii (Nototheniidae). The general morphology of the gills in the two species was slightly different: in T. bernacchii the filament and secondary lamellae were short and thicker, and mitochondria-rich cells were less numerous than in C. hamatus. In the two species the mitochondria-rich cells, distributed in the secondary and primary epithelium, were of the same morphological type, similar to the α-chloride cells of temperate seawater teleosts. The study was carried out to compare the mitochondria-rich cells of Antarctic fishes to the chloride cells of temperate marine teleosts. Immunolocalisation, using a specific antibody to the α-subunit of Na⁺/K⁺-ATPase, was observed in numerous epithelial cells in the interlamellar epithelium and on the secondary lamellae, suggesting an active sodium chloride secretion. Accepted: 16 October 1999     

Recovery from exhausting exercise in an Antarctic fish,Pagothenia borchgrevinki

Summary Pagothenia borchgrevinki, an Antarctic teleost fish was swum to exhaustion and changes in lactic acid levels and blood haematocrit were monitored during recovery. The fish did not perform well at high swimming speeds due to an inability of the white myotomal muscle to produce ATP by anaerobic glycolysis. Consequently, low levels of lactic acid were produced which were fairly rapidly broken down. Haematocrit values were low in non-exercised fish, and these increased by over 100% during exercise, falling back to control levels over many hours. This is probably related to the increased oxygen demand during exercise.     

Ecology of inshore notothenioid fish from the Danco Coast,Antarctic Peninsula

A total of 1,103 inshore notothenioid fish were caught by means of trammel-nets in 4 sites surrounding Cierva Point (Moss Island 1; Moss Island 2; Sterneck Island; Leopardo Island), Danco Coast, West Antarctic Peninsula, during February and March 2000. The families Nototheniidae, Channichthyidae and Bathydraconidae were represented in the samples, Notothenia coriiceps being the dominant fish of the area. Gobionotothen gibberifrons and Trematomus newnesi followed in importance. In general, the fish sampled agreed in terms of number and mass with those of the South Shetland Islands area, except for a marked higher occurrence of G. gibberifrons in the Danco Coast. This supports the hypothesis that the commercial fishery around the South Shetland Islands at the end of the 1970s was responsible for the decrease in the inshore population of G. gibberifrons in that area during the last 17 years. Information on morphometry, reproduction and diet of the fish species caught is provided.     

Ultrastructural observations and pH-measurements on red and white muscle from Antarctic fish

Summary Red and white muscle in the two Antarctic notothenioid fishes Dissostichus mawsoni and Pagothenia borchgrevinki show a rate of postmortem fall of 0.2 pH units per hour, which is close to the rate reported for mammalian muscle at 30°C, but the plateau value is reached several hours earlier in the Antarctic fish, indicating significantly lower stores of initial glycogen. A few particles, most likely representing glycogen, were seen in P. borchgrevinki white muscle and D. mawsoni red muscle, whereas predictably fewer glycogen still was evident in D. mawsoni white muscle. When large numbers of mitochondria and lipid stores were encountered in combination with a small amount of glycogen, we concluded that aerobic metabolism is dominant and that the two species examined would not use white trunk muscle for sustained or slow swimming. Rapid contractions of white trunk muscle as in prey capture or predator evasion are more likely.     

Buoyancy adaptations in a swim-bladderless Antarctic fish

The endemic Antarctic teleosts of the suborder Notothenioidei are bottom dwellers. They lack swim bladders, are heavier than seawater, and feed on or near the bottom. The midwaters surrounding the Antarctic continent are productive and underutilized by fishes. There is an evolutionary trend toward pelagism in some notothenioids. We discovered that the largest Antarctic fish, Dissostichus mawsoni, was neutrally buoyant. Attainment of neutral buoyancy was associated with specializations of the skeletal, integumentary, muscular, and digestive systems. The skeleton had a low mineral content and contained considerable cartilage. Scales were also incompletely mineralized. Static lift was obtained from extensive lipid (mostly triglyceride) deposits. A 2–8 mm subcutaneous lipid layer accounted for 4.7% of the body weight. White muscle also contained much lipid–23% on a dry weight basis, or 4.8% of the body weight. Microscopic examination suggested that the liver was active in lipid metabolism, although it was not an organ of buoyancy. Stellate (perisinusoidal) cells with many lipid droplets were a very prominent cytological component of the liver. These specializations made Dissostichus neutrally buoyant and capable of inhabiting the food-rich Antarctic midwaters.     

Comparison of the solution conformation and dynamics of antifreeze glycoproteins from Antarctic fish

The (1)H- and (13)C-NMR spectra of antifreeze glycoprotein fractions 1-5 from Antarctic cod have been assigned, and the dynamics have been measured using (13)C relaxation at two temperatures. The chemical shifts and absence of non-sequential (1)H-(1)H NOEs are inconsistent with a folded, compact structure. (13)C relaxation measurements show that the protein has no significant long-range order, and that the local correlation times are adequately described by a random coil model. Hydroxyl protons of the sugar residues were observed at low temperature, and the presence of exchange-mediated ROEs to the sugar indicate extensive hydration. The conformational properties of AFGP1-5 are compared with those of the previously examined 14-mer analog AFGP8, which contains proline residues in place of some alanine residues (Lane, A. N., L. M. Hays, R. E. Feeney, L. M. Crowe, and J. H. Crowe. 1998. Protein Sci. 7:1555-1563). The infrared (IR) spectra of AFGP8 and AFGP1-5 in the amide I region are quite different. The presence of a wide distribution of backbone torsion angles in AFGP1-5 leads to a rich spectrum of frequencies in the IR spectrum, as interconversion among conformational states is slow on the IR frequency time scale. However, these transitions are fast on the NMR chemical shift time scales. The restricted motions for AFGP8 may imply a narrower distribution of possible o, psi angles, as is observed in the IR spectrum. This has significance for attempts to quantify secondary structures of proteins by IR in the presence of extensive loops.     

Eco-morphological patterns of the sagitta of Antarctic fish

Morphology and morphometry of the sagittae otolith were studied in pelagic and mesopelagic fish. The shape, margins and rostrum of four groups of otoliths from several species were analyzed: group 1 (pelagic fish associated with the under ice cover N = 42), group 2 (pelagic fish associated with water offshore N = 9), group 3 (mesopelagic fish associated with extensive vertical migration N = 57) and group 4 (mesopelagic fish associated with short vertical migration N = 54). E (maximum width of the sagitta /maximum length of the sagitta %), R (rostrum length (RL)/maximum length of the sagitta %) and S (sulcus area (SS)/otolith area (OS) %) indexes were calculated for each species. Sagittae of pelagic groups (1 and 2) showed the smallest sagitta dimensions in relation to the total length of the fish, in this group the sagitta shape is variable. Sagittae of mesopelagic fish (groups 3 and 4) showed variable shape and edges. The shape in group 4 was polygonal and these species have more width than length. Statistical analysis showed significant differences in the E, R and S indexes. These results were compared with other 19 species, belonging to six families, taken from a publisher-edited literature. E, R and S-values could be used to characterize the sagittae of the Antarctic fish and could be considered as a useful tool for fish ecology studies.     

The oxidation process of Antarctic fish hemoglobins.

Analysis of the molecular properties of proteins extracted from organisms living under extreme conditions often highlights peculiar features. We investigated by UV-visible spectroscopy and X-ray crystallography the oxidation process, promoted by air or ferricyanide, of five hemoglobins extracted from Antarctic fishes (Notothenioidei). Spectroscopic analysis revealed that these hemoglobins share a common oxidation pathway, which shows striking differences from the oxidation processes of hemoglobins from other vertebrates. Indeed, simple exposure of these hemoglobins to air leads to the formation of a significant amount of the low-spin hexacoordinated form, denoted hemichrome. This hemichrome form, which is detected under a variety of experimental conditions, can be reversibly transformed to either carbomonoxy or deoxygenated forms with reducing agents. Interestingly, the spectra of the fully oxidized species, obtained by treating the protein with ferricyanide, show the simultaneous presence of peaks corresponding to different hexacoordinated states, the aquomet and the hemichrome. In order to assign the heme region state of the alpha and beta chains, the air-oxidized and ferricyanide-oxidized forms of Trematomus bernacchii hemoglobin were crystallized. Crystallographic analysis revealed that these forms correspond to an alpha(aquomet)-beta(bishistidyl-hemichrome) state. This demonstrates that the alpha and beta chains of Antarctic fish hemoglobins follow very different oxidation pathways. As found for Trematomus newnesi hemoglobin in a partial hemichrome state [Riccio, A., Vitagliano, L., di Prisco, G., Zagari, A. & Mazzarella, L. (2002) Proc. Natl Acad. Sci. USA99, 9801-9806], the quaternary structures of these alpha(aquomet)-beta(bishistidyl-hemichrome) forms are intermediate between the physiological R and T hemoglobin states. Together, these structures provide information on the general features of this intermediate state.