Carbonic anhydrase activity in tissues of the icefish Chionodraco hamatus and of the red-blooded teleosts Trematomus bernacchii and Anguilla anguilla
Laboratory of General Physiology, Department of Biology, University of Lecce, via Prov.le Monteroni, 73100, Lecce, Italy
*Author for correspondence (e-mail: m.maffia{at}physiology.unile.it)
Accepted August 9, 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The existence of very high CA activity in C. hamatus gills compared with the red-blooded species was investigated further by isolating and characterising the branchial cytosolic CA isoforms. The turnover rate of the C. hamatus isoform was significantly higher than that of T. bernacchii and A. anguilla. The isoforms from both the Antarctic species exhibited lower apparent Km (Km,app) and heat stability than those from A. anguilla. Sensitivity to sulphonamides was similar in all species and was within the range of the mammalian CA II isoform. The branchial CA isoforms of C. hamatus, T. bernacchii and A. anguilla displayed relative molecular masses of 28.9, 29.9 and 31.2 kDa, respectively.
The results suggest that the hemoglobinless teleost possesses a different branchial cytosolic CA isoform from that of red-blooded teleosts.
Key words: Nothotheniodei, Chaenichthyidae, Antarctic teleost, haemoglobinless, pH homeostasis, carbonic anhydrase, blood, gills, Chionodraco hamatus, Trematomus bernacchii, Anguilla anguilla.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The Antarctic icefish of the family Channichthyidae (suborder Nothotheniodei) are a unique example of adult vertebrates lacking haemoglobin and functionally active erythrocytes, and possessing only a small number of erythrocyte-like cells (haematocrit=12 %) (MacDonald and Wells, 1991). Other significant characteristics of their circulatory system include a blood volume and a cardiac output up to sixfold higher than found in other teleosts (Hemmingsen and Douglas, 1970
; Acierno et al., 1995
). The absence of haemoglobin would represent, per se, a dramatic limitation to oxygen transport in the icefish. On the other hand, the very limited number of erythrocyte-like cells (and circulating CA) may compromise CO2/HCO3 equilibria in the blood (Feller and Gerday, 1997
). Owing to the elevated solubility of CO2 in water at low temperatures, and the peculiar characteristics of its circulatory system, CO2 excretion should not be a problem for the icefish. The absence of erythrocytic CA may, however, have an influence on icefish blood acidbase equilibria and pH regulation.
In teleosts, CA has been found in various tissues. It appears to be present in high concentrations in the gills (Rahim et al., 1988; Conley and Mallatt, 1987
), where it plays an important role in osmoregulation, nitrogen (ammonia) excretion, acidbase balance and gas exchange (Henry and Heming, 1998
). Specifically, cytoplasmic CA is believed to function in support of ion transport, and membrane-associated CA is believed to function in facilitated CO2 diffusion. A previous comparative study between two Antarctic species, an icefish Channichthys rhinoceratus and a red-blooded teleost Notothenia magellanica, revealed a much higher CA activity in gill homogenates of the haemoglobinless fish (Feller et al., 1981
). It was hypothesised that this higher activity was related to particular osmoregulatory requirements. No information about its subcellular localisation or biochemical characteristics is, however, available to date. In contrast, in red-blooded teleosts from temperate zones the existence of branchial cytosolic CA isoforms with biochemical characteristics similar to those of mammalian CA II have been demonstrated (Henry et al., 1993
; Sender et al., 1999
).
A potential role for gut CA in acidbase regulation has been suggested in seawater-adapted rainbow trout Oncorhynchus mykiss (Wilson et al., 1996) and in the European eel Anguilla anguilla (Maffia et al., 1996b
). In particular, two CA isoforms have been described in the eel intestine: one is located on the brush border membrane, and mediates bicarbonate absorption from the intestinal lumen, and one is located in the cytosol, and generates HCO3 from metabolic CO2 (Maffia et al., 1996b
).
In the current study, we compared CA activity and distribution in blood, gills, intestine and kidney of the haemoglobinless teleost C. hamatus with those of T. bernacchii, a red-blooded member of the same Antarctic sub-order that shares the same ecotype, and with those of A. anguilla, a teleost from temperate zones. The existence of plasma CA inhibitors in the icefish was also investigated. The cytosolic CA isoforms were purified from the gills of the three species, to compare their molecular mass, kinetic properties, heat stability and sensitivity to sulphonamides, and thereby to investigate potential adaptive mechanisms at a molecular level.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Sample preparation
Blood samples were withdrawn from the caudal vein/artery and added to 0.5 volumes of a cold mixture of (in mmol l1): citric acid 19, sodium acetate 41, D-glucose 81. Blood cells were separated from plasma by centrifugation at 12 000 g for 5 min. In order to collect erythrocyte-free tissues, circulating blood was removed from gills, intestine and kidney by systemic perfusion with heparinised physiological saline for marine teleosts. Animals were killed by a sharp blow to the head and pithed; polyethylene cannulae were then inserted into ventral and dorsal aortas to start the systemic perfusion by means of a peristaltic pump (20 ml min1 kg1 body mass) until erythrocyte-free saline left the circulatory system. Tissues were then frozen in liquid nitrogen and stored at 80°C until experiments. Samples of scraped intestinal mucosa, posterior kidney and gill filaments were dried for 12 h at 120°C for determination of dry mass. Tissue (1 g) was homogenised with a Kinematica Polytron (5 min, maximum rate) in 30 ml of buffer A (in mmol l1): mannitol 300, HepesTris 0.2, adjusted to pH 8.5 with KOH (at 0°C).
Purification of soluble branchial CA
Gill filaments were separated from gill arches, weighed and homogenised in 10 ml of ice-cold phosphate-buffered saline (PBS; 0.92 % NaCl, 0.16 % Na2HPO4·2H2O, 0.02 % NaH2PO4·H2O) g1 wet mass, using a Kinematica Polytron (5 min, maximum speed). The homogenate was centrifuged at 100 000 g for 1 h at 4°C, yeilding a pellet containing cells and membrane fragments and a supernatant containing the cytosolic fraction. Cytosolic branchial CA was purified by affinity chromatography on p-aminomethylbenzene-sulphonamide immobilized on cyanogen-bromide-activated agarose gel (Sigma) (Whitney, 1974). The gel column (17 mmx225 mm) was equilibrated with (in mmol l1): Tris 25, Na2SO4 100, adjusted to pH 8.7 with HCl, and rinsed with Tris 25, NaClO4 300, adjusted to pH 8.7 with HCl; finally the enzyme was eluted in sodium acetate 100, NaClO4 500, pH 5.6. A Pharmacia LKB UV-cord SD was used to follow protein elution at 280 nm. The flow was maintained at 8 ml h1 with a peristaltic pump (Pharmacia LKB Pump-1). The purification was carried out at 2°C. Fractions containing CA activity were collected and concentrated by an ultra filtration cell (Amicon Corp, Lexington, USA) with membrane YM10, under nitrogen pressure (7x105 Pa).
Gel electrophoresis
To test the purity of the enzyme preparation, one-dimensional polyacrylamide (15 %) gel electrophoresis (Bio-Rad Mini-Protean II cell) was performed under denaturating conditions (Laemmli, 1970) in parallel with molecular mass standards (Bio-Rad SDS-Page standards Broad Range) and stained with Coomassie Brilliant Blue R250. The molecular mass of different CA isoforms was calculated from a calibration curve obtained by plotting the relative mobility of standard proteins on the gel against the log of their respective molecular mass for five different SDS-gel electrophoreses.
Determination of CA activity
Electrometric method
CA activity was measured at 0°C, by the electrometric pH method previously described (Wilbur and Anderson, 1948
) as modified by Maffia et al. (1996b
). Briefly, tissue homogenates (50200 µg of protein) and purified CA fractions (0.52 µg of protein) were diluted in 8 ml of buffer A and added to 12 ml of buffer B (in mmol l1): Tris 9.7, Hepes 3.5, pH 8.65. The reaction was started by the addition of 10 ml of CO2-saturated H2O and gassing the assay medium with 5 % CO2, 95 % O2 (CO2 concentration of 3.8 mmol l1). The enzymatic activity, expressed as µmol H+ developed by the hydration of CO2 in excess of a blank sample, was measured by multiplying the change in pH from 8.30 to 8.00 by 2.80 mmol kg1
pH1 (buffer capacity of the incubation medium in that pH range) (Maffia et al., 1996b
) and dividing by the time expressed in min. There were no significant differences in buffer capacity among the different tissues. Protein concentration was measured with Bio-Rad DC protein assay kit, using lyophilised bovine albumin as standard.
Radioactive method
The measurement of CA activity by the electrometric method is technically possible only at a temperature close to 0°C, because of the masking effects of spontaneous CO2 hydration at higher temperatures. Therefore, in order to compare the enzyme activity of the icefish and the eel at their appropriate environmental temperatures, a radioactive assay (Stemler, 1993) was employed to measure the CA activity of the purified branchial isoforms at 0 and 18°C. Durapore membrane filters (GVHP 0.22 µm, Millipore Corporation, Bedford, MA, USA) were used as gas-permeable membranes, while Glass Fiber prefilters (AP25, Millipore Corporation, Bedford, MA, USA) wetted with 0.1 mol l1 NaOH were used as a 14CO2 trap. Purified branchial CA (200 ng) from C. hamatus and A. anguilla were used for each determination. The enzymatic reaction was started by adding NaH14CO3 to the assay mixture containing the enzyme. CA activity was measured at 0 and 18°C for both species by quantifying the amount of 14CO2 trapped within the fiberglass filter at 5, 10 and 20 s from the beginning of the enzymatic reaction. Controls were performed by measuring the 14CO2 trapped within the fiberglass filter in the absence of enzyme.
Inhibition, kinetic analysis and heat stability of carbonic anhydrase
The presence of CA inhibitors in icefish plasma was investigated by adding plasma samples (15400 µl) to the assay buffer. Sensitivity of branchial CA to acetazolamide (ACTZ) and sulphanilamide was examined by measuring CA activity in the presence of increasing concentrations of these inhibitors. Since both ACTZ and sulphanilamide are non-competitive reversible inhibitors, and ACTZ in particular inhibits CA very strongly at nanomolar concentrations, the inhibition constants were determined by plotting the data on an EassonStedman plot. The inhibition constant for ACTZ was calculated as the slope of the straight line described by the following equation:
| (1) |
where I0 is the concentration of inhibitor, i is the fractional inhibition of enzyme activity at a given inhibitor concentration, and E0 is the total concentration of free enzyme in the reaction chamber (Easson and Stedman, 1936). For the determination of kinetic parameters Kcat and apparent Km (Km,app), CA activity was measured by the electrometric method at increasing CO2 concentrations (1, 3, 5, 10, 20, 30, 40, 55, 75 and 100 % CO2, residue O2). Experimental data were fitted to a MichaelisMenten equation with a curve-fitting subroutine in the Graph-Pad software package. Heat stability was evaluated by pre-incubating the purified enzymes for 15 min at increasing temperatures (060°C) and then measuring CA activity by the electrometric method.
Statistical analysis
All data in figures and tables are reported as means ± S.E.M. of at least four different tissue samples. Means were compared by Students t-test while, for multiple comparisons, analysis of variance (ANOVA) was performed. P<0.05 was taken as the fiducial level for statistical significance.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
|
|
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CA activity in blood
Information about blood CO2/HCO3 equilibria in this taxonomic group is scarce, however. In the erythrocytes of red-blooded teleosts, protons derived from CA-dependent CO2 hydration are instantly buffered by haemoglobin (inducing the well known Bohr effect). As a consequence, in the red-blooded species the function of erythrocyte CA is strictly related to the presence of haemoglobin. Our study revealed that CA activity is present in the erythrocyte-like cells of C. hamatus, albeit at much lower levels than that found in the erythrocytes of both the red-blooded species T. bernacchii and A. anguilla, but comparable to that detected in the icefish Channichthys rhinoceratus (Feller et al., 1981). Interestingly, the icefish C. hamatus appears to have preserved a considerable amount of efficient CA inhibitors in its plasma, and these are commonly found in all red-blooded teleosts (Henry et al., 1997
). The possible physiological role of these inhibitors in the blood of a teleost with a very low level of circulating CA is unknown at present, although they may simply be an evolutionary relic. In any case, we consider that no physiologically significant CA activity is detectable in icefish plasma.
CA activity in the gills
Despite the fact that the haemoglobinless fish seem to possess little capacity for anaerobic metabolism, plasma lactic acid concentrations can be similar to other teleosts. That is, owing to the extremely high blood volume, there can be a high total lactic acid content (Feller and Gerday, 1997). It might be hypothesised that, in haemoglobinless teleosts, CA localised on absorptive or secretory epithelia such as the intestine, the kidney and, in particular, the gills, could rapidly supply the blood with the HCO3 required to buffer metabolic acidoses elicited by anaerobic exercise. Indeed, this study revealed a markedly higher CA activity in gills of C. hamatus when compared to the red-blooded species (T. bernacchii and A. anguilla), which confirms previous comparative studies between another icefish species, C. rhinoceratus, and the Antarctic red-blooded species Notothenia rossii (Feller et al., 1981
). We would hypothesise that, in the haemoglobinless teleosts, this epithelium could substitute at least partially for the physiological role of red blood cells in acidbase regulation, by providing a supply of HCO3 to the blood. The higher CA activity in the gills of the icefish cannot, however, be ascribed only to the provision of HCO3 to the blood, because CA is a multifunctional enzyme that is thought to play a role in diverse physiological functions of the fish gill epithelium, including in osmoregulation (Evans et al., 1982
), clearance of the waste products of nitrogen metabolism (Evans and Cameron, 1986
), gas exchange (Randall and Daxboeck, 1984
) and acidbase balance (Heisler, 1984
). Nonetheless, there are good reasons to believe that there are few differences between haemoglobinless and red-blooded teleosts in osmotic water loss or passive salt influx across the gill epithelium. All species that we have considered are seawater animals and the two Antarctic species share the same ecosystem. Indeed, morphometric and physiological studies (Rankin and Tuurala, 1998
) suggest that the only difference in gill structure between the icefish and the red-blooded teleosts is a markedly larger diameter of branchial arteries and marginal channels in the former, which are correlated with the much higher blood flow. Furthermore, since all teleosts under investigation are carnivorous species, nitrogen metabolism should also be somewhat similar, even if the metabolic rates in relation to protein catabolism can be different in temperate compared with Antarctic species.
There is no strong evidence to date for any role of branchial CA in CO2 elimination in teleosts. In red-blooded teleosts, it is generally accepted that erythrocytic CA is the primary and possibly the only site of HCO3 dehydration in the proximity of the respiratory epithelium. A recent review (Henry and Heming, 1998) indicates that gill-membrane-bound CA isoforms could contribute to facilitate CO2 diffusion while soluble (cytoplasmic) CA isoforms should be involved in ion transport. Unfortunately, although our data indicate the presence of a significant amount of membrane-bound CA, we were unable to determine the exact cellular distribution of this isoform. In any case, at low temperatures CO2 solubility in water is significantly increased and, owing to its very large blood volume and cardiac output, icefish could efficiently eliminate the CO2 by storing and transporting it as molecular CO2 in the blood. As a consequence the lack of circulating CA would have no influence on the rates of CO2 excretion at the gill or skin.
For these reasons we hypothesise that the high branchial CA activity found in the gills of C. hamatus, which is mainly from the cytoplasmic isoform, may be related to systemic acidbase regulation. In teleosts, gill CA activity is localised to both pavement and chloride cells (Rahim, 1988; Sender et al., 1999
) where it can rapidly hydrate metabolic CO2 to form HCO3 and H+. HCO3 and a proportion of the protons can be secreted into the blood via basolateral transport systems such as the Cl/HCO3 and the Na+/H+ exchangers. At the apical side, where several teleosts appear to exhibit significant CA activity (Sender et al., 1999
), transporters such as the H+-ATPase and/or the Na+/H+ exchanger could excrete excess protons into the respiratory water. These mechanisms in the icefish gill epithelium would provide it with a means of manipulating plasma bicarbonate levels and buffering blood acidbase disturbances in the absence of erythrocytic CA.
Branchial CA isoforms
Data presented in this study indicate that the putative novel function for CA in the gills of C. hamatus could be performed by an enzymatic isoform with a high turnover rate. Indeed, the catalytic rate (Kcat) measured by the electrometric method (at 0°C) in the branchial C. hamatus isoform was the highest of the investigated species (Table 3). The assay temperature of 0°C is close to the environmental temperature of the Antarctic fish (1.9°C), but is very much lower than the optimal environmental temperature of the European eel. Under such conditions the CA activity measured for the eel gill could, therefore, be significantly underestimated. We used a radiometric method to measure activity at the respective environmental temperatures of the Antartic and temperate species, and confirmed that the activity of the branchial CA in the haemoglobinless teleost at 0°C was higher than that of the eel at 18°C. In terms of activity, there was a significant difference between the CA isoforms isolated from C. hamatus and T. bernacchii, although both displayed a similar substrate affinity, heat stability and molecular mass. At the same time, both the kinetic properties (Fig. 6; Table 3) and the molecular weight (Fig. 5; Table 3) of CA purified from the gills of the Antarctic teleosts were significantly different from those of the enzyme purified from A. anguilla. The CAs purified from C. hamatus and T. bernacchii showed a Kcat value at 0°C that was 2.7- or twofold greater than that found in A. anguilla, respectively (Table 3), a result that is consistent with previous studies comparing homologous enzymes from cold- and warm-adapted species (Somero, 1995). This characteristic may offset the effects of low temperature on the CA activity of Antarctic teleosts as well as sustaining the potentially important role of gill CA in the haemoglobinless fish. The maintenance of an adequate enzyme-substrate affinity could also be an evolutionary strategy to obtain the maximal responsiveness of the enzyme at low temperatures. That the apparent Km for CO2 of the Antarctic teleosts CA isoforms at 0°C was twofold lower than that found in the European eel (Fig. 6; Table 3) agrees with previous results obtained in a comparative analysis of two enzymes, leucine-aminopeptidase and alkaline phosphatase of T. bernacchii and A. anguilla intestine (Maffia et al., 1993
). The sensitivity of inhibition of CA activity to acetazolamide (ACTZ) and sulphanilamide was not significantly different in the three teleost species (Table 3) and was comparable to that measured in the gills of Platichthys flesus (Sender et al., 1999
), rainbow trout Salmo gairdneri (Henry et al., 1993
) and human CAII (Sanyal et al., 1981
). In light of the high sensitivity to sulphonamides and the cytoplasmic localization, the branchial CA isoform of the three teleost species investigated in this study can be considered comparable to the mammalian CAII isoform.
Heat-stability of the Antarctic fish CA isoforms was about 10°C lower than that of the temperate species (Fig. 8). A low heat stability has been observed for trypsin from the Antarctic teleost Pazanothothenia magellanica (Genicot et al., 1988) and for both alkaline phosphatase and Na+-D-glucose cotransporters of T. bernacchii (Maffia et al., 1993
; 1996a
). This behaviour could be ascribed to a reduction in the proportion and/or strength of hydrophobic interactions for the Antarctic fish CA isoform.
An increase in enzyme flexibility resulting from a few amino acid substitutions could be, at least partially, responsible for the very high catalytic rate and substrate affinity of the C. hamatus branchial CA at 0°C. This hypothesis is supported by a partial protein sequence (data not shown) revealing some amino acid substitutions with respect to CA isoforms from Wistar rat brain and the zebrafish Danio rerio, in regions distant from the active site, which seem to be highly conserved in all species.
A dissimilar protein structure or glycosylation could cause the different electrophoretic migration of the enzymes from C. hamatus and A. anguilla (Fig. 5). As determined by SDS-PAGE, the molecular mass of approx. 29 kDa of the C. hamatus isoform indicates that a novel enzyme variant with high turnover rate is located in the respiratory epithelium of the icefish.
In conclusion, comparing the three species, it appears that C. hamatus branchial CA displays two different aspects of evolutionary adaptation; the first is related to cold-adaption and the second is associated with the absence of erythrocytes. The possible role of this enzyme in rapidly supplying blood with bicarbonate may be a compensatory mechanism for blood pH regulation in the absence of erythrocytic CA.
Detailed information on the cell expression of this cytosolic CA isoform and eventually of other membrane-bound CAs, as well as their exact localization in the different cell types of the gill epithelium, could give a more complete picture of the role of this enzyme in icefish gill, clarifying the overall function of the respiratory epithelium in these teleosts. Finally, sequence analysis of the novel CA isoform, comparative studies with other enzyme isoforms, and site-directed mutagenesis experiments, could give us information on the molecular basis of the evolved adaptation mechanisms to survival in a cold environment.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Acierno, R., Macdonald, J. A., Agnisola, C. and Tota, B. (1995). Blood volume in the haemoglobinless Antarctic teleost Chionodraco hamatus (Lonnberg). J. Exp. Zool. 272, 407409.
Acierno, R., Maffia, M., Rollo, M. and Storelli, C. (1997). Buffer capacity in the blood of the hemoglobinless Antarctic fish Chionodraco hamatus. Comp. Biochem. Physiol. 118A, 989992.
Conley, D. M. and Mallatt, J. (1987). Histochemical localization of Na+-K+ ATPase and carbonic anhydrase activity in gills of 17 fish species. Can. J. Zool. 66, 23982405.
Easson, L. H. and Stedman, E. (1936). The absolute activity of choline-esterase. Proc. R. Soc. Lond. B 121, 142164.
Evans, D. H. and Cameron, J. N. (1986). Gill ammonia transport. J. Exp. Zool. 239, 1723.
Evans, D. H., Claiborne, J. B., Farmer, L., Mallery, C. and Krasny, E. K. (1982). Fish gill ionic transport: methods and models. Biol. Bull. Mar. Biol. Lab. Woods Hole 163, 108130.
Feller, G. and Gerday, C. (1997). Adaptations of the haemoglobinless antarctic icefish (Channichthyidae) to hypoxic tolerance. Comp. Biochem. Physiol. 118A, 981987.
Feller, G., Pequeux, A. and Hamoir, G. (1981). La presence danhydrase carbonique chez 2 poissons de larchipel des Kerguelen, Channichthys rhynoceratus exempt dhemoglobine et Notothenia magellanica de formule sanguine normale. CR Acad. Sci. Paris 293, 395397.
Feller, G., Poncin, A., Aittaleb, M., Schyns, R. and Gerday, C. (1994). The blood proteins of the Antarctic icefish Channichthys rhinoceratus: biological significance and purification of the two main components. Comp. Biochem. Physiol. 109B, 8997.
Genicot, S., Feller, G. and Gerday, C. H. (1988). Trypsin from Antarctic fish (Pazanothothenia magellanica Forster) as compared with trout (Salmo gairdneri) trypsin. Comp. Biochem. Physiol. 90B, 601609.
Heisler, N. (1984). Acidbase regulation in fishes. In Fish Physiology, vol. XA (ed. W. S. Hoar and D. J. Randall), pp. 315401. New York: Academic Press.
Hemmingsen, E. A. and Douglas, E. L. (1970). Respiratory characteristics of the haemoglobin-free fish Chaenocephalus aceratus. Comp. Biochem. Physiol. 39, 773742.
Henry, R. P. and Heming, T. A. (1998). Carbonic anhydrase and respiratory gas exchange. In Fish Physiology Haemoglobin and Respiration, vol. 17 (ed. S. F. Perry and B. L. Tufts), pp. 75111. San Diego: Academic Press.
Henry, R. P. and Swenson, E. R. (2000). The distribution and physiological significance of carbonic anhydrase in vertebrate gas exchange organs. Resp. Physiol. 121, 112.[Medline]
Henry, R. P., Gilmour, K. M., Wood, C. M. and Perry, S. F. (1997). Extracellular carbonic anhydrase activity and carbonic anhydrase inhibitors in the circulatory system of fish. Physiol. Zool. 70, 650659.[Medline]
Henry, R. P., Tufts, B. L. and Boutilier, R. G. (1993). The distribution of carbonic anhydrase type I and II isozymes in lamprey and trout: possible co-evolution with erythrocyte chloride/bicarbonate exchange. J. Comp. Physiol. 163B, 380388.
Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680685.[Medline]
MacDonald, J. A. and Wells, R. M. G. (1991). Viscosity of body fluids from Antarctic Notothenioid fish. In The Biology of Antarctic Fishes (ed. G. di Prisco, B. Maresca and B. Tota), pp. 163178. Berlin: Springer Verlag.
Maffia, M., Acierno, R., Cillo, E. and Storelli, C. (1996a). Na+-D-glucose cotransport by intestinal BBMVs of the Antarctic fish Trematomus bernacchii. Am. J. Physiol. 271, R1576R1583.
Maffia, M., Acierno, R., Deceglie, G., Vilella, S. and Storelli, C. (1993). Adaptation of intestinal cell membrane enzymes to low temperature in the Antarctic teleost Pagothenia bernacchii. J. Comp. Physiol. 163B, 265270.
Maffia, M., Trischitta, F., Lionetto, M. G., Storelli, C. and Schettino, T. (1996b). Bicarbonate absorption in eel intestine: evidence for the presence of membrane-bound carbonic ahydrase on the brush border membranes of the enterocyte. J. Exp. Zool. 275, 365373.[Medline]
Perry, S. F. and Laurent, P. (1990). The role of carbonic anhydrase in carbon dioxide excretion, acidbase balance and ionic regulation in aquatic gill breathers. In Animal Nutrition and Transport Processes. 2. Transport, Respiration and Excretion: Comparative and Environmental Aspects, vol VI, Comparative Physiology (ed. J. P. Truchot and B. Lahlou), pp. 3957. Basel: Karger.
Rahim, S. M., Delaunoy, J. P. and Laurent, P. (1988). Identification and immunocytochemical localization of two different carbonic anhydrase isoenzymes in teleostean fish erythrocytes and gill epithelia. Histochemistry 89, 451459.[Medline]
Randall, D. J. and Daxboeck, C. (1984). Oxygen and carbon dioxide transfer across fish gills. In Fish Physiology, vol. XA (ed. W. S. Hoar and D. J. Randall), pp. 263314. New York: Academic Press.
Rankin, J. C. and Tuurala, H. (1998). Gills of Antarctic fish. Comp. Biochem. Physiol. 119A, 149163.
Sanyal, G., Pessah, N. I. and Maren, T. H. (1981). Kinetics and inhibition of membrane bound carbonic anhydrase from canine renal cortex. Biochem. Biophys. Acta 657, 128137.[Medline]
Sender, S., Bottcher, K., Cetin, Y. and Gros, G. (1999). Carbonic anhydrase in the gills of seawater and freshwater-acclimated flounders Platichthys flesus: purification, characterization, and immunohistochemical localization. J. Histochem. Cytochem. 47, 4350.
Sly, W. S. and Peiyi, Y. H. (1995). Human carbonic anhydrases and carbonic anhydrase deficiencies. Annu. Rev. Biochem. 64, 375401.[Medline]
Somero, G. N. (1995). Proteins and temperature. Annual Rev. Physiol. 57, 4368.[Medline]
Stemler, A. (1993). An assay for carbonic anhydrase activity and reactions that produce radiolabeled gases or small uncharged molecules. Anal. Biochem. 210, 328331.[Medline]
Tesch, F. W. (1977). Blood. In The Eel (ed. P. H. Greenwood), pp. 3841. London: Chapman and Hall.
Wells, R. M. G., Summers, G., Beard, L. A. and Grigg, G. C. (1988). Ecological and behavioural correlates of intracellular buffering capacity in the muscles of Antarctic fishes. Polar. Biol. 8, 321325.
Whitney, P. L. (1974). Affinity chromatography of carbonic anhydrase. Anal. Biochem. 57, 467476.[Medline]
Wilbur, K. M. and Anderson, N. G. (1948). Electrometric and colorimetric determination of carbonic anhydrase. J. Biol. Chem. 176, 147154.
Wilson, R. W., Gilmour, K. M., Henry, R. P. and Wood, C. M. (1996). Intestinal base excretion in the seawater-adapted rainbow trout: a role in acidbase balance? J. Exp. Biol. 199, 23312343.