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The blood of Antarctic icefishes (family Channichthyidae, suborder Notothenioidei) is completely devoid of hemoglobin. Icefishes have developed compensatory adaptations that reduce oxygen demand and enhance oxygen transport. Oxygen delivery to tissues occurs by carrying the gas physically dissolved in the plasma. To evaluate the evolutionary pathway leading to the icefish hemoglobinless phenotype, the adult and embryonic/juvenile gene complexes from a closely related, red-blooded notothenioid species were isolated and characterized. The hybridization pattern of notothenioid adult globin cDNAs showed that the genomes of three icefish species retain transcriptionally inactive alpha1-globin-related DNA sequences, which are identical truncated variants of the alpha1-globin gene of the red-blooded fish, containing part of intron 2, all of exon 3, and the 3'-untranslated region. The icefish genomes have no beta-globin genes. Furthermore, Southern blots of genomic DNA from red- and white-blooded (two species) notothenioids, probed with fragments of the genes flanking the ends of the embryonic/juvenile complex, indicated that icefishes have also lost embryonic/juvenile globin genes. It is proposed that inability to express hemoglobin arose from a single, large-scale deletional event, which removed all icefish globin genes with the exception of the 3' end of alpha1.

Tetrameric hemoglobins are the most widely used systems in studying protein cooperativity. Allosteric effects in hemoglobins arise from the switch between a relaxed (R) state and a tense (T) state occurring upon oxygen release. Here we report the 2.0-A crystal structure of the main hemoglobin component of the Antarctic fish Trematomus newnesi, in a partial hemichrome form. The two alpha-subunit iron atoms are bound to a CO molecule, whereas in the beta subunits the distal histidine residue is the sixth ligand of the heme iron. This structure, a tetrameric hemoglobin in the hemichrome state, demonstrates that the iron coordination by the distal histidine, usually associated with denaturing states, may be tolerated in a native-like hemoglobin structure. In addition, several features of the tertiary and quaternary organization of this structure are intermediate between the R and T states and agree well with the R --> T transition state properties obtained by spectroscopic and kinetic techniques. The analysis of this structure provides a detailed pathway of heme-heme communication and it indicates that the plasticity of the beta heme pocket plays a role in the R --> T transition of tetrameric hemoglobins.

Psychrobacter sp. TAD1 is a psychrotolerant bacterium from Antarctic frozen continental water that grows from 2 to 25 degrees C with optimal growth rate at 20 degrees C. The new isolate contains two glutamate dehydrogenases (GDH), differing in their cofactor specificities, subunit sizes and arrangements, and thermal properties. NADP+-dependent GDH is a hexamer of 47 kDa subunits and it is comparable to other hexameric GDHs of family-I from bacteria and lower eukaria. The NAD+-dependent enzyme, described in this communication, has a subunit weight of 160 kDa and belongs to the novel class of GDHs with large size subunits. The enzyme is a dimer; this oligomeric arrangement has not been reported previously for GDH. Both enzymes have an apparent optimum temperature for activity of approximately 20 degrees C, but their cold activities and thermal labilities are different. The NAD+-dependent enzyme is more cold active: at 10 C it retains 50% of its maximal activity, compared with 10% for the NADP+-dependent enzyme. The NADP+-dependent enzyme is more heat stable, losing only 10% activity after heating for 30 min, compared with 95% for the NAD+-dependent enzyme. It is concluded that in Psychrobacter sp. TAD1 not only does NAD+-dependent GDH have a novel subunit molecular weight and arrangement, but that its polypeptide chains are folded differently from those of NADP+-dependent GDH, providing different cold-active properties to the two enzymes.

Hemoglobin function is modulated by several non-heme ligands; among these effectors, organic phosphates generally bind to heterotropic sites with a one-to-one stoichiometry. The phosphate binding site of human hemoglobin is located at the interface between the two beta chains. An additional binding site for polyanions has been studied at the molecular level (Tamburrini, M., A. Riccio, M. Romano, B. Giardina, and G. di Prisco. 2000. Eur. J. Biochem. 267:6089-6098) in the hemoglobins of the south polar skua (Catharacta maccormicki). It is formed by a cluster of six positive charges of both alpha chains (Val-1, Lys-99, Arg-141); the two Lys-99alpha have an essential role in the site structure. The present investigation, carried out on skua deoxyhemoglobins by using a molecular dynamics approach, confirms the structural feasibility of the additional site, possibly having the role of an entry-leaving site, and leads to the proposal of a novel migration pathway for phosphate along the central cavity of hemoglobin from one binding site to the other, occurring according to the hypothesis of a site-site migratory mechanism, which may assign a functional role to the central cavity. The role of Lys-99alpha was further confirmed by molecular dynamics experiments on the mutant Lys-99alpha-->Ala in which, at the end of the simulation, the phosphate was external to the additional site.

Liganded and unliganded forms of two Antarctic fish haemoglobins, from Trematomus newnesi and T. bernacchii, have been crystallized in low-salt media using polyethylene glycol as precipitant. In particular, crystals of air-exposed T. newnesi carbomonoxy haemoglobin were found to be isomorphous to the crystals grown in high-salt media. Preliminary X-ray analysis of the diffraction data revealed that the beta-haem iron of this haemoglobin is in the haemichrome state, with both the proximal and distal histidyl residues linked to the iron. This is the first crystallization of a haemichrome intermediate of a vertebrate haemoglobin.

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion-exchange chromatography. The oxygen-binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5-8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co-operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co-operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co-operativity, and displayed a normal Bohr effect; the addition of chloride increased co-operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino-acid sequences of the alpha and beta chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.

Hemoglobins extracted from fishes that live in temperate waters show little or no dissociation even in the liganded form, unlike human hemoglobin (HbA). To establish whether cold adaptation influences the tendency to dissociate, the dimer-tetramer association constants (L(2,4)) of the carbonmonoxy derivatives of representative hemoglobins from two Antarctic fishes, Trematomus newnesi (Hb1Tn) and Trematomus bernacchii (Hb1Tb), were determined by analytical ultracentrifugation as a function of pH in the range 6.0-8.6 and compared to HbA. HbA is more dissociated than fish hemoglobins at all pH values and in particular at pH 6.0. In contrast, both fish hemoglobins are mostly tetrameric over the whole pH range studied. The extent of hydrophobic surface area buried at the alpha(1)beta(2) interface upon association of dimers into tetramers and the number of hydrogen bonds formed are currently thought to play a major role in the stabilization of the hemoglobin tetramer. These contributions were derived from the X-ray structures of the three hemoglobins under study and found to be in good agreement with the experimentally determined L(2,4) values. pH affects oxygen binding of T. bernacchii and T. newnesi hemoglobins in a different fashion. The lack of a pH effect on the dissociation of the liganded proteins supports the proposal that the structural basis of such effects resides in the T (unliganded) structure rather than in the R (liganded) one.

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 Q(10), achieved by increased k(cat) and modulation of S(0.5), 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.

The amino-acid sequence and the oxygen-binding properties of the two haemoglobins of the Antarctic seabird south polar skua have been investigated. The two haemoglobins showed peculiar functional features, which were probably acquired to meet special needs in relation to the extreme environmental conditions. Both haemoglobins showed a weak alkaline Bohr effect which, during prolonged flight, may protect against sudden and uncontrolled stripping of oxygen in response to acidosis. We suggest that a weak Bohr effect in birds may reflect adaptation to extreme life conditions. The values of heat of oxygenation suggest different functional roles of the two haemoglobins. The experimental evidence suggests that both haemoglobins may bind phosphate at two distinct binding sites. In fact, analysis of the molecular models revealed that an additional phosphate binding site, formed by residues NA1alpha, G6alpha and HC3alpha, is located between the two alpha chains. This additional site may act as an entry/leaving site, thus increasing the probability of capturing phosphate and transferring it to the main binding site located between the two beta chains by means of a site-site migratory mechanism, thereby favouring the release of oxygen. It is suggested that most haemoglobins possess an additional phosphate binding site, having such a role in oxygen transport.

The Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1 contains two distinct glutamate dehydrogenases (GDH), each specific for either NADP+ or NAD+. This feature is quite unusual in bacteria, which generally have a single GDH. NADP+-dependent GDH has been purified to homogeneity and the gene encoding GDH has been cloned and expressed. The enzyme has a hexameric structure. The amino acid sequence determined by peptide and gene analyses comprises 447 residues, yielding a protein with a molecular mass of 49 285 Da. The sequence shows homology with hexameric GDHs, with identity levels of 52% and 49% with Escherichia coli and Clostridium symbiosum GDH, respectively. The coenzyme-binding fingerprint motif GXGXXG/A (common to all GDHs) has Ser at the last position in this enzyme. The overall hydrophilic character is increased and a five-residue insertion in a loop between two alpha-helices may contribute to the increase in protein flexibility. Psychrobacter sp. TAD1 GDH apparent temperature optimum is shifted towards low temperatures, whereas irreversible heat inactivation occurs at temperatures similar to those of E. coli GDH. The catalytic efficiency in the temperature range 10-30 degrees C is similar or lower than that of E. coli GDH. Unlike E. coli GDH the enzyme exhibits marked positive cooperativity towards 2-oxoglutarate and NADPH. This feature is generally absent in prokaryotic GDHs. These observations suggest a regulatory role for this GDH, the most crucial feature being the structural/functional properties required for fine regulation of activity, rather than the high catalytic efficiency and thermolability encountered in several cold-active enzymes.

As new structural data have become available, somewhat contrasting explanations of the Root effect in fish haemoglobins (Hb) have been provided. Hb 1 of the Antarctic fish Trematomus newnesi has a nearly pH-independent oxygen affinity, in spite of 95 % sequence identity with Hb 1 of Trematomus (previously named Pagothenia) bernacchii that has a strong Root effect. Here, the 2.2 A R-state structure of Trematomus newnesi Hb 1 is presented. The structure is similar to that of Root effect fish Hbs from Spot and T. bernacchii, suggesting that the differences in the pH dependence cannot be related to the modulation of the R-state. In comparison to T. bernacchii Hb 1, the role of the three mutations Thr41 (C6)alpha-->Ile, Ala97 (G3)alpha-->Ser and His41 (C7)beta-->Tyr at the alpha1beta2-interface is discussed., (Copyright 1998 Academic Press.)

In the framework of a study on molecular adaptations of the oxygen-transport and storage systems to extreme conditions in Antarctic marine organisms, we have investigated the structure/function relationship in Emperor penguin (Aptenodytes forsteri) myoglobin, in search of correlation with the bird life style. In contrast with previous reports, the revised amino acid sequence contains one additional residue and 15 differences. The oxygen-binding parameters seem well adapted to the diving behaviour of the penguin and to the environmental conditions of the Antarctic habitat. Addition of lactate has no major effect on myoglobin oxygenation over a large temperature range. Therefore, metabolic acidosis does not impair myoglobin function under conditions of prolonged physical effort, such as diving.

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 alpha and beta 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.

In the present report, we describe a novel aspartic proteinase from the liver of two Antarctic fish species. The nucleotide sequences of the cDNA obtained from the two fishes show 90% identity with each other but only 58% identity with aspartic proteinases from other sources. Sequence analysis shows features for the Antarctic enzymes which are not present in related enzymes of other organisms.

TAD1 is a psychrophilic strain isolated from continental frozen water in Antarctica. Study of aspartate transcarbamylase in the bacterium shows an impressive activity of this enzyme at low temperature. At 0 degree C, its activity is up to 26% of its maximal activity observed at 30 degrees C. In comparison with the Escherichia coli enzyme, some of its kinetic properties suggest that this high activity at low temperature results from an increased catalytic efficiency. This property might result from a discrete modification localized at the catalytic site, since this psychrophilic enzyme is as stable as its Escherichia coli homologue at high temperature.

The icefishes of the Southern Ocean (family Channichthyidae, suborder Notothenioidei) are unique among vertebrates in their inability to synthesize hemoglobin. We have shown previously (Cocca, E., Ratnayake-Lecamwasam, M., Parker, S. K., Camardella, L., Ciaramella, M., di Prisco, G., and Detrich, H. W., III (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 1817-1821) that icefishes retain inactive genomic remnants of adult notothenioid alpha-globin genes but have lost the gene that encodes adult beta-globin. Here we demonstrate that loss of expression of the major adult alpha-globin, alpha1, in two species of icefish (Chaenocephalus aceratus and Chionodraco rastrospinosus) results from truncation of the 5' end of the notothenioid alpha1-globin gene. The wild-type, functional alpha1-globin gene of the Antarctic yellowbelly rockcod, Notothenia coriiceps, contains three exons and two A + T-rich introns, and its expression may be controlled by two or three distinct promoters. Retained in both icefish genomes are a portion of intron 2, exon 3, and the 3'-untranslated region of the notothenioid alpha1-globin gene. The residual, nonfunctional alpha-globin gene, no longer under positive selection pressure for expression, has apparently undergone random mutational drift at an estimated rate of 0.12-0.33%/million years. We propose that abrogation of hemoglobin synthesis in icefishes most likely resulted from a single mutational event in the ancestral channichthyid that deleted the entire beta-globin gene and the 5' end of the linked alpha1-globin gene.

Reverse transcriptase-mediated PCR has been used to isolate two distinct metallothionein (MT) cDNA species from RNA extracted from icefish liver, namely MT-I and MT-II. Northern blot analysis with these cDNA species revealed that significant endogenous levels of MT mRNA were present in liver tissues of normal animals despite the fact that no MT protein could be found accumulating in the same tissue. However, multiple injections of CdCl2 induced high levels of both MT mRNA and MT protein. Sequence analysis of the cDNA species that were present after cadmium injection revealed the presence of both isoforms. Quantification of the MT-I and MT-II transcripts from normal and heavy-metal-treated fish showed an alteration in the ratio of the MT isoform transcripts. Endogenous transcripts consisted mostly of MT-II, whereas the MT-I transcript was preferentially accumulated only in response to the cadmium salt. The protein encoded by each cDNA isoform was isolated from the heavy-metal-treated fish and the availability of the specific MT mRNA for translation was demonstrated by translation in vitro. These results show that: (1) there is a discrepancy between the significant endogenous levels of MT mRNA and the absence of MT protein; (2) the accumulation of MT in icefish liver can be triggered by heavy metals; (3) genes encoding distinct MT isoforms are differentially regulated by heavy metals.

The hematological properties and the oxygen-transport system of the antarctic fish Pleuragramma antarcticum were investigated. Most blood parameters are at the lower end of the range of values known for red-blooded antarctic fish, suggesting a link with the sluggish mode of life of this species. P. antarcticum is the only species of the family Nototheniidae and of the suborder Notothenioidei having three major hemoglobins, which were isolated and fully characterized. The complete amino acid sequence of the alpha- and beta-globin chains was determined. The three hemoglobins showed strong Bohr and Root effects, and their oxygen-binding properties were differently regulated by temperature. None of the three hemoglobins of P. antarcticum can be considered as evolutionary (or larval) remnants. Therefore, this oxygen-transport system is one of the most specialized ever found in fish. The data suggest a strong relationship between hematological/biochemical adaptation and life style.

Glucose-6-phosphate dehydrogenase (G6PD) and L-glutamate dehydrogenase (GDH) from Antarctic fish were isolated and characterized. G6PD was purified from the erythrocytes of red-blooded Dissostichus mawsoni and from the colorless blood of the icefish Chionodraco hamatus. Structural and functional characterization showed that the two enzymes do not differ significantly from each other. GDH was purified from the liver of the icefish Chaenocephalus aceratus. As in other fish ODHs, it showed a marked preference for NAD-. The amino acid sequence of the active-site peptide is virtually identical to that of other fish and vertebrate counterparts. Although the basic structural features of the Antarctic enzymes are similar to those of mesophilic organisms, some catalytic and thermodynamic properties make the Antarctic enzymes more suited to cold-adapted organisms.

Studies of the hemoglobin system of fish of the suborder Notothenioidei have been extended to non-Antarctic species of Pseudaphritis urvillii and Notothenia angustata. The two species belong to families that were the first to diverge within the suborder. The degree of amino acid sequence identity with Antarctic notothenioids and other non-Antarctic fish species is analyzed with respect to phyletic and ecological diverence.

Amino acid sequences of alpha- and beta-chains of human hemoglobin and of hemoglobins of coelacanth and 24 teleost fish species, including 11 antarctic and two temperate Notothenioidei, were analyzed using maximum parsimony. Trees were derived for the alpha- and beta-chains separately and for tandemly arranged sequences, using the human and coelacanth sequences as outgroups in all analyses. The topologies of the trees of the alpha- and beta-chains are highly congruent and indicate a specific pattern of gene duplications and gene expression of teleost hemoglobins which has not yet been investigated into more detail. The Notothenioid fish generally contain a single major hemoglobin and often a second minor component. The alpha- and beta-chains of the major components form a monophyletic group in all investigated trees, with the nonantarctic Pseudaphritis as their sister taxon. The minor chains also are a monophyletic group and form an unresolved cluster with the major chains and the hemoglobins of tuna and red gurnard. The Notothenioid families Nototheniidae and Bathydraconidae appear to be paraphyletic.

Icefish (family Channichthyidae, suborder Nothothenioidei) are a group of Antarctic fish that have evolved unique phenotypes in order to adapt to the environment in which they live. Besides the lack of haemoglobin and the drastic reduction in the number of erythrocyte-like cells, another striking feature of the icefish is that their liver is devoid of metallothionein. These cysteine-rich heavy-metal-binding proteins are usually present in large amounts in a large variety of organisms, from bacteria to mammals. Despite the failure to detect appreciable levels of metallothionein in icefish liver, a cDNA encoding metallothionein was produced from total RNA by reverse transcriptase PCR. The icefish metallothionein showed high percentage identity with metallothionein from Trematomus bernachii, a red-blooded Antarctic fish in which a normal content of hepatic metallothionein was found. Steady-state mRNA levels were assessed in fish liver by high-stringency hybridization of the metallothionein probe with total RNA. The results showed that icefish livers retain large amounts of untranslated metallothionein mRNA. The stability of the icefish transcript might be correlated with the lack of specific motifs in the untranslated 3' ends of mRNA.

The pH dependence of CO binding kinetics to Chelidonichthys kumu hemoglobin (Hb) and human adult Hb has been investigated between pH 2.0 and 9.0 at 20 degrees C. For both Hbs, CO binding kinetics is characterized by two proton-linked transitions, with different pKa values for alpha- and beta-chains in C. kumu Hb, leading to a relevant functional kinetic heterogeneity at most pH values. On the other hand, in human adult Hb the CO binding does not display a functional heterogeneity. Lowering the pH from 9 to 6 brings about a decrease of the CO binding rate constants, to a different extent for human adult Hb and the two chains of C. kumu Hb. Further lowering the pH from 6 to 2 induces an enhancement of CO binding rate constants, probably related to the protonation of proximal HisF8 Nepsilon atom and the cleavage (or severe weakening) of the HisF8-Fe bond. The presence of physiological concentrations of ATP (approximately 3 mM) affects the pH dependence of CO binding kinetics to C. kumu. Moreover, the effect of temperature (between 8 degrees C and 38 degrees C) on CO binding kinetics has been investigated in the absence of ATP at different pH values. These results allow to interpret the functional kinetic heterogeneity of C. kumu Hb on the basis of different regulatory aspects in the alpha- and beta-subunits, as suggested by structural considerations.

Concerning the number and type of the hemoglobin components, the moray Muraena helena is characterized by three different phenotypes whose frequencies are nearly identical. Thus, the cathodal component is present in all individuals, whereas one or both of two anodal components may be present in the same phenotype. These components have been separated by chromatography. The oxygen binding properties of the purified hemoglobin components have been studied in the absence and presence of saturating concentrations of ATP or GTP and as a function of pH. The cathodal component shows an intrinsic O2 affinity four times higher than that of both anodal components, a very small Bohr effect and a significant decrease in O2 affinity upon addition of ATP and GTP (three and four times respectively with respect to stripped conditions), the latter being more effective than the former over the entire pH range examined. The anodal components do not appear functionally distinguishable and show the presence of an enhanced Bohr effect (Root effect) that is under the strict control of nucleotide triphosphates ATP, GTP, which, unlike in the cathodic component, exert the same effect on oxygen affinity. The complete sequence of the beta chains of the cathodal and of one of the anodal components have been determined. The possible molecular basis of these different functional characteristics are discussed in the light of the globin sequence and of those amino acid residues which are known to be responsible of hemoglobin functional behaviour.

As in other fish, the cathodic hemoglobin of the eel Anguilla anguilla is considered to play an important role in oxygen transport under hypoxic and acidotic conditions. In the absence of phosphates this hemoglobin shows a reverse Bohr effect and high oxygen affinity, which is strongly modulated over a side pH range by GTP (whose concentration in the red blood cells varies with ambient oxygen availability). GTP obliterates the reverse Bohr effects in the cathodic hemoglobin. The molecular basis for the reverse Bohr effect in fish hemoglobins has remained obscure due to the lack of structural data. We have determined the complete amino acid sequence of the alpha and beta chains of the cathodic hemoglobins of A. anguilla and relate it to the oxygen equilibrium characteristics. Several substitutions in crucial positions are observed compared with other hemoglobins, such as the replacement of the C-terminal His of the beta chain of Phe (that suppresses the alkaline Bohr effect) and of residues at the switch region between alpha and beta subunits (that may alter the allosteric equilibrium, thus causing the high intrinsic oxygen affinity and low cooperativity). The residues binding organic phosphate in the beta cleft of fish hemoglobins are conserved, which explains the strong effect of GTP on oxygen affinity and suggests that these residues contribute to the reverse Bohr effect in the absence of alkaline Bohr groups. Moreover, His beta 143 that is considered to be responsible for the reverse Bohr effect in human and tadpole Hbs is replaced by Lys.

Glucose-6-phosphate dehydrogenase (G6PD) has been purified from the blood of two Antarctic teleost species, i.e., from the erythrocytes of Dissostichus mawsoni (family Nototheniidae), and from the plasma and cells of haemoglobinless Chionodraco hamatus (family Channichthyidae). The specific activities in haemolysates of Antarctic blood cells appear higher than that of a lysate of human erythrocytes. The two Antarctic enzymes have an apparent subunit molecular mass slightly higher than that of human G6PD; the electrophoretic behaviour on cellulose acetate is similar. Both Antarctic enzymes are irreversibly heat inactivated through a biphasic process. Km for glucose-6-phosphate (G6P) does not vary significantly with temperature, whereas Km for NADP increases at increasing temperature, kcat increases with temperature, with a break point at 35 degrees C (in human G6PD, the break point is at 15 degrees C). Thermodynamic and kinetic characterisation indicate that the catalytic performance of the enzyme of cold-adapted fish, at temperatures typical of their habitat, is more efficient than that displayed by G6PD from a temperature organism.

Alone among piscine taxa, the antarctic icefishes (family Channichthyidae, suborder Notothenioidei) have evolved compensatory adaptations that maintain normal metabolic functions in the absence of erythrocytes and the respiratory oxygen transporter hemoglobin. Although the uniquely "colorless" or "white" condition of the blood of icefishes has been recognized since the early 20th century, the status of globin genes in the icefish genomes has, surprisingly, remained unexplored. Using alpha- and beta-globin cDNAs from the antarctic rockcod Notothenia coriiceps (family Nototheniidae, suborder Notothenioidei), we have probed the genomes of three white-blooded icefishes and four red-blooded notothenioid relatives (three antarctic, one temperate) for globin-related DNA sequences. We detect specific, high-stringency hybridization of the alpha-globin probe to genomic DNAs of both white- and red-blooded species, whereas the beta-globin cDNA hybridizes only to the genomes of the red-blooded fishes. Our results suggest that icefishes retain inactive genomic remnants of alpha-globin genes but have lost, either through deletion or through rapid mutation, the gene that encodes beta-globin. We propose that the hemoglobinless phenotype of extant icefishes is the result of deletion of the single adult beta-globin locus prior to the diversification of the clade.

The functional properties of the single haemoglobin (Hb) of Emperor penguin (Aptenodytes forsteri) have been investigated at different temperatures as a function of proton and organic phosphate concentration. The complete amino acid sequence has been established. Comparison with that of human HbA shows 12 substitutions in the contact regions of alpha beta dimers. In addition to overall similarities shared with most of the avian Hbs previously described, this Hb shows significant differences, which could be related to the peculiar behaviour of this penguin. In particular we may consider that: (1) the shape of the Bohr effect curve seems well adapted for gas exchange during very prolonged dives, preserving penguin Hb from a sudden and not controlled stripping of oxygen; (2) the very minor enthalpy change observed at lower pH could be an example of molecular adaptation, through which oxygen delivery becomes essentially insensitive to exposure to the extremely low temperatures of the environment. Moreover, the small alkaline Bohr effect has been found to be only chloride-linked, since the pH dependence of the oxygen affinity is totally abolished in the absence of this ion. These functional characteristics are discussed on the basis of the primary structure of alpha and beta-chains.

Antarctic fish of the family Nototheniidae usually have a single major hemoglobin (Hb 1), often a second, minor component (Hb 2, about 5% of the total), and traces of another component (Hb C, less than 1%). These are functionally similar Bohr and Root effect hemoglobins. All species of other highly endemic fish families so far investigated also have one single major hemoglobin. The hematological features of the nototheniid Trematomus newnesi are remarkably different. It is the only Antarctic species in which Hb 1 and Hb 2 display only a very weak Bohr effect and no Root effect. Perhaps consequentially, Hb C (the only component showing regulation of oxygen binding by protons and other effectors) is not present in traces but accounts for 20-25% of the total. The primary structure of the three hemoglobins of T. newnesi and of Root effect HbC present in trace amounts in another nototheniid (Pagothenia bernacchii) is discussed in relationship with oxygen binding and in terms of molecular and stereochemical models. The hemoglobin multiplicity, the oxygen binding features of Hb 1 and Hb 2, and the presence of functionally distinct components, thus reveal that the oxygen transport of T. newnesi has unique characteristics.

The blood of the teleost Chelodonichthys kumu, living in the temperate waters of New Zealand, contains a single hemoglobin. The complete amino acid sequence of the alpha and beta chain has been established. The presence of a reactive Cys in the external position beta CD8(49) causes polymerisation through intermolecular disulfide bridges between beta chains, with no alteration of functional features. C. kumu Root-effect hemoglobin displays very low or no subunit co-operativity in the physiological pH range. Kinetic experiments on the oxygen dissociation and binding of carbon monoxide show a marked, pH-dependent functional heterogeneity of the two chains, which contributes to the observed reduction of co-operativity. In contrast, kinetic heterogeneity was not observed in the process of CO dissociation, indicating that functional differences between the subunits are detectable only for the dynamic ligand association pathway. The allosteric effector, ATP, seems to increase the pKa of the proton-linked effect on the slow-reacting subunit, affecting the quaternary equilibrium through stabilisation of the T state at lower pH, rather than enhancing the functional heterogeneity itself. In position E11 of both chains, Val (usually present at the distal side of the heme), is substituted by Ile. Although this residue has been shown not to significantly alter ligand binding to the alpha chain, to some extent it can perturb the access of oxygen to the beta chain. Thus, this substitution may be the main reason for subunit functional heterogeneity.

The blood of the teleost Notothenia angustata contains a major hemoglobin (Hb 1, over 95% of the total), accompanied by a minor component (Hb 2). The two hemoglobins have identical beta chains and differ in their alpha chains. The primary structure of both hemoglobins has been established through the elucidation of the complete amino acid sequence of the three chains. The study of the oxygen-binding properties shows that Hb 1 displays the Bohr and Root effects and has high affinity for organic phosphates. N. angustata belongs to the family Nototheniidae, suborder Notothenioidei. Unlike the vast majority of nototheniid species, which live in isolation in the Antarctic Ocean and have developed cold adaptation, N. angustata inhabits the waters of southern New Zealand and is not cold adapted. Although some hematological parameters typically favour oxygen transport in a temperate environment, the hemoglobin multiplicity and structural and functional features closely resemble those of the Antarctic species of the same family and suborder. Thus, N. angustata may be considered as a link between temperate and Antarctic habitats. The hypothetical separation history of N. angustata from the Antarctic species of the same family is discussed in the light of the present findings.

1. A zinc-binding protein has been isolated and purified from the liver of the icefish Chionodraco hamatus. 2. The icefish Zn-protein has characteristics distinct from those of metallothionein. 3. The amino acid composition shows a low content of cysteine and a high content of glutamate and aspartate. 4. No metallothionein has been detected in the extracts from icefish liver.

1. Bathydraco marri Norman is a cold-adapted Antarctic teleost (Family: Bathydraconidae), living preferably at depths between 400 and 1200 m. 2. The blood of this species contains a single hemoglobin, in which oxygen binding is pH-regulated (Bohr and Root effects). 3. The complete amino acid sequence of the alpha and beta chains of the hemoglobin of B. marri has been elucidated.

The extensive in vivo conversion of haemoglobin to the carbon monoxide derivative has no discernible effect on the survival of the red-blooded Antarctic fish Pagothenia bernacchii. Analysis of caudally sampled blood of cannulated specimens revealed that reconversion of carbon-monoxy haemoglobin to oxyhaemoglobin was complete within 48 hours. Thus, under stress-free conditions, haemoglobin is not necessary for survival of P. bernacchii. Red-blooded Antarctic fishes can carry oxygen necessary for routine delivery dissolved in plasma, in a similar way to the haemoglobinless Channichthyidae, although they lack the morphological and physiological adaptations which allow the latter to prosper without any haemoglobin.

The Antarctic fish Pagothenia bernacchii has one major haemoglobin, Hb1 (over 95% of the total blood content). Hb1 has a strong alkaline Bohr effect and at low pH exhibits the reduced ligand affinity and co-operativity that comprise the Root effect. We have determined the complete amino acid sequence of P. bernacchii Hb1 and also the structure of its carbonmonoxy derivative by X-ray crystallography, to a resolution of 2.5 A. The crystallographic R-factor of the refined structure is 18%. The three-dimensional structure of this fish haemoglobin is similar to that of human haemoglobin A, with a root-mean-square difference in main-chain atom positions of 1.4 A after superimposition of the two structures, despite only 48% homology of their amino acid sequences (including insertion of a single residue in the CD region of the fish alpha-chain). Large structural differences occur only at the N and C termini of both the alpha- and beta-chains. Neither these nor other smaller structural differences provide any obvious explanation of the Root effect of this or other fish haemoglobins.

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.

Evolution has adopted different strategies to solve the problem of transporting oxygen to respiring tissues, according to needs dictated by the environment. A thermodynamic analysis of haemoglobins of organisms living in extreme polar environments (mammals and fish) provides elegant examples of such adaptations.

The blood of the teleost Cygnodraco mawsoni, of the endemic Antarctic family Bathydraconidae, contains a major hemoglobin (Hb 1), accompanied by a minor component (Hb 2, about 5% of total). The two hemoglobins have identical alpha chains and differ by the beta chain. The complete amino acid sequence of the three chains has been elucidated, thus establishing the primary structure of both hemoglobins. The sequences show a 53-65% identity with non-Antarctic poikilotherm fish species; on the other hand, a very high degree of similarity (83-88%) has been found between Hb 1 and the major component of another Antarctic species of a different family. The hemoglobin functional properties relative to oxygen binding have been investigated in intact erythrocytes, 'stripped' hemolysate and purified components of C. mawsoni. The hemoglobins display the Bohr and Root effects, indicating fine regulation of oxygen binding by pH and by the physiological effectors organic phosphates.

1. Notothenia coriiceps neglecta is a cold-adapted notothenioid teleost, widely distributed in the Antarctic waters. 2. In comparison with fishes from temperate waters, the blood of this teleost contains a reduced number of erythrocytes and concentration of hemoglobin; the erythrocytes contain two hemoglobins, Hb1 and Hb2, respectively accounting for approximately 90, and 5% of the total. 3. The two components differ by the alpha chain; the amino acid sequence of the beta chain in common to the two hemoglobins has been established, thus completing the elucidation of the primary structure of the major component Hb 1.

1. The blood of Notothenia coriiceps neglecta (a cold-adapted notothenioid fish, widely distributed in Antarctic waters, and characterized by a relatively low content of erythrocytes and hemoglobin), contains two hemoglobin components, Hb 1 and Hb 2; the amino acid sequences of the beta chain of Hb 1 and Hb 2 are identical. 2. The amino acid sequence of the alpha chain of Hb 2 has been established, thus completing the elucidation of the primary structure of the two hemoglobins.

Human erythrocyte glucose-6-phosphate dehydrogenase was purified to homogeneity by a simplified procedure, consisting of 2',5'-ADP-Sepharose affinity chromatography, followed by Sephadex G-100 gel filtration. The carboxy-terminal region of the protein was identified by carboxypeptidase digestion: the sequence -Lys-Leu-COOH was found instead of the reported -Gly-COOH, thus showing identity with the carboxy-terminal sequence of glucose-6-phosphate dehydrogenase from human leukocytes and platelets. In addition, the carboxyl-terminal peptide was isolated from a tryptic digest of the protein and sequenced. The sequence is: Trp-Val-Asp-Pro-His-Lys-Leu.

Antarctic fishes live at a constant temperature of -1.8 degrees C, in an oxygen-rich environment. In comparison with fishes that live in temperate or tropical waters, their blood contains less erythrocytes and hemoglobin. A study was initiated on the structure and function of Antarctic fish hemoglobin. The erythrocytes of the Antarctic benthic teleost Notothenia coriiceps neglecta, of the family Nototheniidae, have been shown to contain two hemoglobins, accounting for about 90% and 5% of the total content. These hemoglobins have been isolated, and obtained in crystalline form. They are tetramers and contain two pairs of globin chains. The globin chains of each hemoglobin have been purified and characterised. The two hemoglobins appear to have one of the two globin chains in common. The Root and Bohr effects have been investigated in erythrocytes, 'stripped' hemolysates and pure hemoglobins, indicating that the functional properties are finely regulated by pH and allosteric effectors.

The blood of the Antarctic fish Notothenia coriiceps neglecta contains two hemoglobins, Hb 1 and Hb 2, which have a beta-chain in common. We have elucidated the primary structure of the beta-chain (146 residues) and of the alpha-chains (142 residues) of the two hemoglobins. The two alpha-chains differ from each other by 51 residues; in comparison with globin sequences of temperate fishes, the alpha-chain of Hb 1 is more similar to that of bluefin tuna than to the alpha-chain of Hb 2 of the same species.

The complete amino acid sequence of the alpha chain of the main hemoglobin of the Antarctic fish Notothenia coriiceps neglecta (family Nototheniidae) has been determined. It consists of 142 residues; an acetylated seryl residue is at the amino terminal. The molecular mass is 15,519 Da. In comparison with alpha-chain sequences of non-Antarctic poikilothermic fish hemoglobins, the homology appears to be significantly lower than that existing among the latter species. A higher homology has been found with the alpha-chain sequence of the non-poikilothermic bluefin tuna.

Structural differences between crystalline mitochondrial and nuclear glutamate dehydrogenases from ox liver have been detected by immunological techniques. Antisera prepared against each enzyme precipitate both glutamate dehydrogenases; upon immunodiffusion, the antiserum against the nuclear enzyme gives a line of incomplete identity with the two antigens, whereas the antiserum against the mitochondrial enzyme gives a line of complete identity. Fractionation of the antibodies contained in each antiserum by means of an immunoadsorbent, to which the nuclear or the mitochondrial enzyme has been covalently linked, shows that nuclear glutamate dehydrogenase (GDH) contains specific antigenic determinants as well as determinants common to the mitochondrial enzyme, whereas the latter appears to have no antigenic portions which are not present in the nuclear antigen, in accord with the results of immunodiffusion. The antibodies against determinants common to both enzymes precipitate and inhibit them, whereas the specific anti-nuclear GDH antibodies precipitate but do not inhibit the nuclear antigen.