Structure of haptoglobin and the haptoglobin-hemoglobin complex by electron microscopy

The human serum protein, haptoglobin, forms a stable, irreversible complex with hemoglobin. Haptoglobin is composed of two H chains, which are connected via two smaller L chains to give a protein of 85,000 Mr. In the complex, each H chain binds an alpha beta dimer of hemoglobin for a total molecular weight of 150,000. The scanning transmission electron microscope has been used to derive new information about the shape and structure of haptoglobin and hemoglobin, and about their relative orientation in the complex. The micrographs of negatively stained images show that haptoglobin has the shape of a barbell with two spherical head groups, which are the H chains. These are connected by a thin filament with a central knob, which corresponds to the L chains. The overall length of the molecule is about 124(+/- 8) A and the interhead distance is 87 (+/- 7) A. In the haptoglobin-hemoglobin complex, the head groups are ellipsoidal and under optimal staining conditions bilobal . Thus, the alpha beta dimers are binding to the H chains, but off the long axis of the barbell by 127 degrees in a trans configuration. This angle considerably restricts the region on the surface of the H chain structure that can contain the hemoglobin binding site. The interhead group distance for complex is 116.5(+/- 6.3) A or 30 A greater than for haptoglobin. The N terminus of the beta chain was located on the trans off-axis configured barbell structure of complex by using a hemoglobin that was crosslinked between the alpha beta dimers in the region of the beta N terminus. The distances and angles that are measured on the micrographs for the native and crosslinked complex molecules permit the directions of two of the alpha beta dimer ellipsoid axes to be assigned. Taken together, these data provide an approximate relative orientation for the binding of the alpha beta dimer to the H chain of haptoglobin.     

Studies on free or haptoglobin-bound hemoglobin and derivatives (semihemoglobins and porphyrinated semihemoglobins). Some aspects of their peroxidatic activity.

The peroxidatic activity of hemoglobin (Hb) is known to be enhanced when this hemoprotein is bound to haptoglobin (Hp). The peroxidatic reaction (H2O2, guaiacol as donor) has been kinetically studied (Steady-state) in the presence of free or rabbit-haptoglobin bound human hemoglobin and some of its derivatives, all in ferricyano-form. With free Hb+ CN, we observed linearity of Lineweaver and Burk plots in a wide range of concentrations, the donor's behaviour was therefore assumed to obey the Michaelis-Menten mechanism. When Hp-Hb+ CN is the enzyme, the donor's behaviour is more complicated, analysis shows the existence of two kinds of donor's binding sites. The possibility whether this behaviour might correspond to the intrinsic properties of Hb chains, as revealed after combination with Hp, was examined. The peroxidatic activity of free and Hp-bound alpha and beta chains of Hb were studied. The alpha chains of Hb combine with Hp whereas the beta chains fail to do so. In order to make useful comparisons, the peroxidatic activity of Hp-bound alpha and beta chains were studied by the use of Hp-semihemoglobin complexes where the semihemoglobins carried heme on only one type of chain (alpha or beta). Results did not show an evident correlation between the activities of the two free or bound types of chains and those of the two classes of binding sites revealed in Hp-Hb+ CN. Moreover, it appeared that the heme-free complementary chain might influence the activity of the heme-carrying alpha or beta chain in semihemoglobins and Hp-semihemoglobin complexes. The binding or protoporphyrin on free and Hp-bound semihemoglobins leads to species which exhibit structures close to that of Hb and Hp-Hb complex respectivley. Results of studies on these derivatives brought up new interesting data : when the porphyrin ring alone is bound to the heme deficient chains (alpha or beta), in Hp-semihemoglobin complexes, the same peculiar behaviour, already observed with Hp-Hb complex, is found again. The structural implications of these results are discussed.     

Molecular cloning of human haptoglobin cDNA: evidence for a single mRNA coding for alpha 2 and beta chains

Human haptoglobin (Hp) is a plasma glycoprotein composed of alpha and beta polypeptide chains that are covalently associated by disulfide bonds. It had been suggested that alpha and beta polypeptides could be synthesized via a common precursor polypeptide. We report the molecular cloning of DNA complementary to human Hp mRNA. One of the clones, pULB1148, carries a full length copy coding for both alpha 2 and beta polypeptides. In vitro translation of human liver mRNA hybridizing with this cDNA gives a protein mol. wt. of 49000 daltons. The sequence of the alpha 2 beta cDNA shows the presence of a single Arg residue between Gln 142 of the alpha 2 chain and Ileu 1 of the beta chain. With a few minor exceptions, the DNA sequence fits the previously published amino acid sequences. The differences are the presence of an Asp residue at position 52 of alpha 2 instead of Asn, the existence in beta of only one Lys residue between Gly 65 and the following Gln, the presence of Ser and Cys at positions 218-219 instead of Cys-Ser, and of Asp residues at positions 205 and 235 instead of Asn.     

Human haptoglobin structure and function--a molecular modelling study

Hemoglobin is the most prominent protein in blood, transporting O(2) and facilitating reactive oxygen and nitrogen species detoxification. Hemoglobin metabolism leads to the release of extra-erythrocytic hemoglobin, with potentially severe consequences for health. Extra-erythrocytic hemoglobin is complexed to haptoglobin for clearance by tissue macrophages. The human gene for haptoglobin consists of three structural alleles: Hp1F, Hp1S and Hp2. The products of the Hp1F and Hp1S alleles differ by only one amino acid, whereas the Hp2 allele is the result of a fusion of the Hp1F and Hp1S alleles, is present only in humans and gives rise to a longer alpha-chain. Haptoglobin consists of a dimer of alphabeta-chains covalently linked by a disulphide bond between the Cys15 residue of each alpha-chain. However, the presence of the Hp1 and Hp2 alleles in humans gives rise to HPT1-1 dimers (covalently linked by Cys15 residues), HPT1-2 hetero-oligomers and HPT2-2 oligomers. In fact, the HPT2 variant displays two free Cys residues (Cys15 and Cys74) whose participation in intermolecular disulphide bonds gives rise to higher-order covalent multimers. Here, the complete modelling of both haptoglobin variants, together with their basic quaternary structure arrangements (i.e. HPT1 dimer and HPT2 trimer), is reported. The structural details of the models, which represent the first complete view of the molecular details of human haptoglobin variants, are discussed in relation to the known haptoglobin function(s).     

Simple high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin

Haptoglobin is an acute-phase protein and its plasma levels increase consistently in response to infection and inflammation. Some evidence has demonstrated that haptoglobin is involved in the immune responses. In this study, we established a novel high-performance liquid chromatographic purification procedure for porcine plasma haptoglobin. The procedure required an ammonium sulfate fractionation and a HPLC Superose 12 gel-permeation chromatography. Purified porcine haptoglobin possessed one heavy (β) and light chain (α) on sodium dodecyl sulfate–polyacrylamide gel electrophoresis, under reducing conditions, with a M_r (molecular mass) of about 42 000 and 14 000 for heavy (β) and light chains (α), respectively. Although the N-terminal amino acid sequence of porcine heavy chain of haptoglobin has never been reported previously, the analyses of N-terminal amino acid sequence showed a great sequence similarity to that of human and other animal species. In addition, Western blot using our specific antibody prepared against porcine M_r 42 000 chain did react with human haptoglobin and likewise, the antibody against human haptoglobin also cross-reacted with purified porcine M_r 42 000 chain. Thus, it confirmed that the identity of the porcine protein purified from our procedures was as haptoglobin.     

Quantitative determination of haptoglobin glycoform variants in psoriasis

Haptoglobin is an acute phase glycoprotein, secreted by hepatocytes and other types of cells including keratinocytes. Haptoglobin has been suggested to impair the immune response, inhibit gelatinases in the extracellular matrix and promote angiogenesis, but its role in psoriasis is obscure to date. Changes in haptoglobin glycan structure were observed in several diseases. The aim of this study was to investigate whether haptoglobin displays glycan variations in psoriasis. We found that the pattern of plasma haptoglobin glycoforms, following two-dimensional electrophoresis, exhibited significant quantitative differences in spot intensities between patients and controls. Quantitative and qualitative differences in glycan mass, between patients and controls, were found by mass spectrometry of glycopeptides from tryptic digests of protein isolated from both patients and controls. The number of distinct fucosylated glycoforms of peptides NLFLNHSENATAK and MVSHHNLTTGATLINEQWLLTTAK was higher in patients than in controls, but no fucosylated glycan was detected on peptide VVLHPNYSQ-VDIGLIK in either case. The number of peptides with distinct triantennary and tetraantennary glycans was higher in patients than in controls. Abundance or structure of specific glycans, which are present in haptoglobin from patients and are different or missing in normal haptoglobin, might be associated with disease activity.     

Proteomic Analysis of Haptoglobin and Amyloid A Protein Levels in Patients with Vivax Malaria

Advancements in the field of proteomics have provided great opportunities for the development of diagnostic and therapeutic tools against human diseases. In this study, we analyzed haptoglobin and amyloid A protein levels of vivax malaria patients with combinations of depletion of the abundant plasma proteins, 2-dimensional gel electrophoresis (2-DE), image analysis, and mass spectrometry in the plasma between normal healthy donors and vivax malaria patients. The results showed that the expression level of haptoglobin had become significantly lower or undetectable in the plasma of vivax malaria patients due to proteolytic cleavage when compared to healthy donors on 2-DE gels. Meanwhile, serum amyloid A protein was significantly increased in vivax malaria patient''s plasma with high statistical values. These 2 proteins are common acute phase reactants and further large scale evaluation with a larger number of patient''s will be necessary to establish the possible clinical meaning of the existential changes of these proteins in vivax malaria patients. However, our proteomic analysis suggests the feasible values of some plasma proteins, such as haptoglobin and serum amyloid A, as associating factor candidates for vivax malaria.     

Proteomics of ischemia and reperfusion injuries in rabbit myocardium with and without intervention by an oxygen-free radical scavenger

A brief period of ischemia followed by timely reperfusion may lead to prolonged, yet reversible, contractile dysfunction (myocardial stunning). Damage to the myocardium occurs not only during ischemia, but also during reperfusion, where a massive release of oxygen-free radicals (OFR) occurs. We have previously utilized 2-DE and MS to define 57 protein spot changes during brief ischemia/reperfusion (15 min ischemia, 60 min reperfusion; 15I/60R) injury in a rabbit model (White, M. Y., Cordwell, S. J., McCarron, H. C. K., Prasan, A. M. et al., Proteomics 2005, 5, 1395-1410) and shown that the majority of these occur because of physical and/or chemical PTMs. In this study, we subjected rabbit myocardium to 15I/60R in the presence of the OFR scavenger N-(2-mercaptopropionyl) glycine (MPG). Thirty-seven of 57 protein spots altered during 15I/60R remained at control levels in the presence of MPG (15I/60R + MPG). Changes to contractile proteins, including myosin light chain 2 (MLC-2) and troponin C (TnC), were prevented by the addition of MPG. To further investigate the individual effects of ischemia and reperfusion, we generated 2-DE gels from rabbit myocardium subjected to brief ischemia alone (15I/0R), and observed alterations of 33 protein spots, including 18/20 seen in both 15I/60R-treated and 15I/60R + MPG-treated tissue. The tissue was also subjected to ischemia in the presence of MPG (15I/0R + MPG), and 21 spot changes, representing 14 protein variants, remained altered despite the presence of the OFR scavenger. These ischemia-specific proteins comprised those involved in energy metabolism (lactate dehydrogenase and ATP synthase alpha), redox regulation (NADH ubiquinone oxidoreductase 51 kDa and GST Mu), and stress response (Hsp27 and 70, and deamidated alpha B-crystallin). We conclude that contractile dysfunction associated with myocardial stunning is predominantly caused by OFR damage at the onset of reperfusion, but that OFR-independent damage also occurs during ischemia. These ischemia-specific protein modifications may be indicative of early myocardial injury.     

Relevance of the amino acid conversions L144R (Zaragoza) and L159P (Zavalla) in the apolipoprotein A-I binding site for haptoglobin

The high-density lipoprotein apolipoprotein A-I (ApoA-I) stimulates the enzyme lecithin-cholesterol acyltransferase (LCAT) in the reverse cholesterol transport pathway. Two ApoA-I variants, Zaragoza (L144R) and Zavalla (L159P), are associated with low levels of HDL-cholesterol but normal LCAT activity. Haptoglobin interacts with ApoA-I, impairing LCAT stimulation. Synthetic peptides matching the haptoglobin-binding site of native or variant ApoA-I (native, P2a; variants, Zav-pep and Zar-pep) bound haptoglobin with different activity: Zar-pep>P2a>Zav-pep. They also differently rescued LCAT in vitro activity in the presence of haptoglobin (P2a=Zar-pep>Zav-pep). Therefore, both amino acid conversions affect haptoglobin binding and LCAT regulation. We highlight the role of haptoglobin in LCAT regulation in subjects with ApoA-I variants.     

Canine haptoglobin: a unique haptoglobin subunit arrangement.

1. Isolated canine haptoglobin behaved identically to the alpha 2 beta 2 structure typical of human haptoglobin type 1-1 on alkaline polyacrylamide gel electrophoresis and on gel filtration. 2. In the presence of urea or sodium dodecyl sulphate canine haptoglobin dissociated into alpha beta subunits that separated into alpha and beta chains after reduction with 2-mercaptoethanol. 3. Compositional analysis identified one less half-cystine in canine alpha chain when compared to human alpha 1 chain. 4. These results provide evidence that there is no inter alpha chain disulphide in canine haptoglobin comparable to the alpha 1 20-alpha 1 20 disulphide in human haptoglobin that links the two alpha beta subunits.     

Lethal perinatal osteogenesis imperfecta due to the substitution of arginine for glycine at residue 391 of the alpha 1(I) chain of type I collagen

A baby with the lethal perinatal form of osteogenesis imperfecta was shown to have a structural defect in the alpha 1(I) chain of type I procollagen. Normal and mutant alpha 1(I) CB8 cyanogen bromide peptides, from the helical part of the alpha 1(I) chains, were purified from bone. Amino acid sequencing of tryptic peptides derived from the mutant alpha 1(I) CB8 peptide showed that the glycine residue at position 391 of the alpha 1(I) chain had been replaced by an arginine residue. This substitution accounted for the more basic charged form of this peptide that was observed on two-dimensional electrophoresis of the collagen peptides obtained from the tissues. The substitution was associated with increased enzymatic hydroxylation of lysine residues in the alpha 1(I) CB8 and the adjoining CB3 peptides but not in the carboxyl-terminal CB6 and CB7 peptides. This finding suggested that the sequence abnormality had interfered with the propagation of the triple helix across the mutant region. The abnormal collagen was not incorporated into the more insoluble fraction of bone collagen. The baby appeared to be heterozygous for the sequence abnormality and as the parents did not show any evidence of the defect it is likely that the baby had a new mutation of one allele of the pro-alpha 1(I) gene. The amino acid substitution could result from a single nucleotide mutation in the codon GGC (glycine) to produce the codon CGC (arginine).     

The serum proteome of Equus caballus

We constructed a reference two-dimensional protein map for horse (Equus caballus) serum. The serum proteins were separated by two-dimensional electrophoresis (2-DE); 29 different gene products were identified. Proteins represented by 25 spots/spot groups were identified by tandem nanoelectrospray mass spectrometry (MS), four by matrix-assisted laser desorption ionization time-of-flight (TOF) MS and one was sequenced by TOF-TOF technology. The identities of four proteins were deduced by similarity to the human plasma protein database. In selected cases, i.e. the immunoglobulins, immunoblotting with specific antibodies provided additional information about the respective proteins. Albumin was detected as the full-length protein and as fragments of various sizes. Spots representing products of different mass and charge were also detected for alpha1-antitrypsin, haptoglobin and transthyretin. Thus, despite the fact that the Equus caballus genome is incompletely characterized, we were able to identify almost all moderate to high abundance proteins stained in the serum 2-DE pattern.     

Binding of human hemoglobin and its polypeptide chains with haptoglobin coupled to an agarose matrix.

The interactions of human haptoglobin covalently linked to agarose with human hemoglobin and with p-chloromercuribenzoic-acid-treated alpha and beta chains (alpha* and beta* chains) were studied by flow chromatography and equilibrium binding. The results indicate that in solid state, haptoglobin maintains the same binding characteristics as in solution, the order of binding affinities being: hemoglobin greater than alpha* chain greater than beta* chain. The study of the binding parameters of the alpha* chain shows an heterogeneity of binding sites on the haptoglobin and an average affinity constant Ka of 3.6 X 10(4)l/mol.     

Multi-component immunoaffinity subtraction chromatography: an innovative step towards a comprehensive survey of the human plasma proteome

In order to discover novel protein markers indicative of disease processes or drug effects, the proteomics technology platform most commonly used consists of high resolution protein separation by two-dimensional electrophoresis (2-DE), mass spectrometric identification of proteins from stained gel spots and a bioinformatic data analysis process supported by statistics. This approach has been more successful in profiling proteins and their disease- or treatment-related quantitative changes in tissue homogenates than in plasma samples. Plasma protein display and quantitation suffer from several disadvantages: very high abundance of a few proteins; high heterogeneity of many proteins resulting in long charge trains; crowding of 2-DE separated protein spots in the molecular mass range between 45-80 kD and in the isoelectric point range between 4.5 and 6. Therefore, proteomic technologies are needed that address these problems and particularly allow accurate quantitation of a larger number of less abundant proteins in plasma and other body fluids. The immunoaffinity-based protein subtraction chromatography (IASC) described here removes multiple proteins present in plasma and serum in high concentrations effectively and reproducibly. Applying IASC as an upfront plasma sample preparation process for 2-DE, the protein spot pattern observed in gels changes dramatically and at least 350 additional lower abundance proteins are visualized. Affinity-purified polyclonal antibodies (pAbs) are the immunoaffinity reagents used to specifically remove the abundant proteins such as albumin, immunoglobulin G, immunoglobulin A, transferrin, haptoglobin, alpha-1-antitrypsin, hemopexin, transthyretin, alpha-2-HS glycoprotein, alpha-1-acid glycoprotein, alpha-2-macroglobulin and fibrinogen from human plasma samples. To render the immunoaffinity subtraction procedure recyclable, the pAbs are immobilized and cross-linked on chromatographic matrices. Antibody-coupled matrices specific for one protein each can be pooled to form mixed-bed IASC columns. We show that up to ten affinity-bound plasma proteins with similar solubility characteristics are eluted from a mixed-bed column in one step. This facilitates automated chromatographic processing of plasma samples in high throughput, which is desirable in proteomic disease marker discovery projects.     

Amino acid sequence and disulfide-bridge location of canine haptoglobin.

The complete amino acid sequence and disulfide-bridge location of canine haptoglobin (Hp) were determined by analyzing various fragments produced chemically and/or enzymatically. Canine Hp consists of two light (L) and two heavy (H) chains with 83 and 245 amino acid residues, respectively. It has three potential oligosaccharide-binding sequences, Asn-X-Ser/Thr, one in the L chain and two in the H chain. All of them are glycosylated. Comparison of the amino acid sequences between canine Hp and human Hp shows 68 and 85% homology for L chains and H chains, respectively. About 20% of the canine L chain still retains a carboxyl-terminal arginine residue, which is completely removed during maturation in human L-chain. The half-cystine residue at position 15 in the L chain, which participates in the inter-L chain disulfide bridging in human Hp, has been replaced by a leucine residue in canine Hp. Therefore, an LH unit in canine Hp may be joined to another LH unit by a noncovalent (mainly hydrophobic) interaction to form the complete molecule. The disulfide bridges in canine Hp link Cys-34L to Cys-68L, Cys-72L to Cys-105H, Cys-148H to Cys-179H, and Cys-190H to Cys-220H, as in the case of human Hp.     

Reference maps of mouse serum acute-phase proteins: changes with LPS-induced inflammation and apolipoprotein A-I and A-II transgenes

We present reference maps of the mouse serum proteome (run under reducing and non-reducing conditions), from control animals, from mice injected with lipopolysaccharide (LPS) to induce systemic inflammation, and from mice transgenic for human apolipoproteins A-I and A-II. Seventy-seven spots/spot chains from the reducing gels were identified by HPLC MS/MS, representing 28 distinct proteins, including a species-specific protease inhibitor, contrapsin, and high levels of carboxylesterase. The concentrations of acute-phase reactants were monitored for 96 h after LPS challenge. The greatest changes (four-fold 48 h after LPS administration) were observed for haptoglobin and hemopexin. Orosomucoid/alpha(1)-acid glycoprotein and apolipoprotein A-I increased steadily, to 50-60% above baseline at 96 h from stimulation. In mice transgenic for human apolipoprotein A-I the levels of expression of orosomucoid/alpha(1)-acid glycoprotein, alpha(1)-macroglobulin, esterase, kininogen and contrapsin were altered compared to knockout mice lacking apolipoprotein A-I. In contrast, except for the presence of apolipoprotein A-II, no statistically significant difference was observed in mice transgenic for human apolipoprotein A-II.     

Peptide inhibitors of CDK2-cyclin A that target the cyclin recruitment-site: structural variants of the C-terminal Phe

A focused series of octapeptides based on the lead compound H-His-Ala-Lys-Arg-Arg-Leu-Ile-Phe-NH(2) 1, in which the C-terminal phenylalanine residue was replaced by alpha and/or beta-modified variants, was synthesized using solid-phase chemistry. Both the L-threo-beta-hydroxy-phenylalanine (beta-phenylserine, Pse) and (2S)-phenylalaninol derivatives, as competitive binders at the cyclin-recruitment site, displayed potent inhibitory activity towards the CDK2-cyclin A complex. Unexpectedly, the D-threo-Pse derivatives also showed inhibitory activity.     

触珠蛋白研究进展

触珠蛋白是一种酸性糖蛋白,属急性期反应蛋白之一,由于所含轻链类型的不同,触珠蛋白具有遗传多态性,触珠蛋白的合成和降解主要在肝脏进行,并受细胞因子、前列腺素、激素等的调节,触珠蛋白具有广泛的生物学功能,可能是一种重要的调节蛋白.     

Non-helical sequences of rabbit collagen. Correlation with antigenic determinants detected by rabbit antibodies in homologous regions of rat and calf collagen.

Non-helical peptide fragments were isolated from rabbit skin collagen after cleavage of alpha chains with cyanogen bromide and proteases. Determination of their amino acid sequence indicated a length of 9, 16 and 25 amino acid residues for the non-helical sequences located in the N-terminal region of alpha2 and alpha1 chain and in the C-terminal region of alpha1 chain, respectively. The C-terminal sequence Tyr-Tyr hitherto considered as the genuine end of collagen alpha1 chain is in part of rabbit collagen extended by two residues, alanine and arginine. Rabbit collagen may differ considerably in its non-helical sequences from other vertebrate collagens, particularly in the C-terminal part. Some but not all of these differences are clustered in areas occupied by antigenic determinants which are recognized in the antibody response of rabbits to rat or calf collagen. On the other hand, a high homology to rabbit collagen, e.g. in the N-terminal region of rat collagen alpha1 chain or calf collagen alpha2 chain, probably prevents immunological recognition by the rabbit. The degree of foreignness alone, however, may not necessarily determine whether a particular non-helical area is able to express immunogenic activity.     

Cold-stable microtubules from Antarctic fishes contain unique alpha tubulins

The cytoplasmic microtubules of Antarctic fishes assemble from their tubulin subunits at physiological body temperatures in the range -2 to +2 degrees C. Our objective is to determine the structural features that enhance the assembly of Antarctic fish tubulins at low temperatures. Here we compare the structures of tubulin subunits from three Antarctic fishes (Notothenia gibberifrons, Notothenia coriiceps neglecta, and Chaenocephalus aceratus), from three temperate fishes (the dogfish shark Mustelus canis, the channel catfish Ictalurus punctatus, and the goosefish Lophius americanus), and from a mammal (the cow Bos taurus). When reduced, carboxymethylated, and examined by polyacrylamide gel electrophoresis, multiple alpha chains were observed in tubulins from the Antarctic fishes, the catfish, and the goosefish; dogfish and bovine alpha tubulins migrated as single components on this gel system. Prominent in the Antarctic fish tubulins was an alpha variant that migrated more rapidly than the bovine alpha chain; smaller amounts of a rapidly migrating alpha chain were also present in catfish and goosefish tubulins. The beta tubulins of the fishes, with the exception of the goosefish, resolved into major and minor variants with mobilities similar to those of beta 1 and beta 2 tubulins from bovine brain. Peptide mapping demonstrated that the alpha tubulins of Antarctic fishes were similar in structure, yet differed from the alpha chains of the dogfish and the cow (which, in turn, were similar to each other). In contrast, the beta tubulins from these organisms gave peptide patterns of near identity. Finally, the alpha chains of native tubulins from N. coriiceps neglecta and the cow differed in the sensitivity of their C-terminal domains to digestion by subtilisin. These results demonstrate that the alpha tubulins of Antarctic fishes (but not their beta chains) differ structurally from those of temperate fishes and a mammal.