- Email: [email protected]
Red cell carbonic anhydrase levels in flounders, Platichthys flesus L., from salt water and fresh water
Comp. Biochera. Physiol., 1976, Vol. 55B, pp. 399 to 401. Pergamon Press. Printed in Great Britain
RED CELL CARBONIC ANHYDRASE LEVELS IN FLOUNDERS, P L A T I C H T H Y S FLESUS L., FROM SALT WATER AND FRESH WATER NICHOLAS CARTER,* JENNIFER AUTON* AND PAUL DANDO~" *Department of Biochemistry, London Hospital Medical College, London, E1 2AD and t Marine Biological Research Association, Citadel Hill, Plymouth, Devon, U.K.
(Received 20 February 1976) AMlract--1. Carbonic anhydrase levels in flounder red cells were unchanged by adaptation to salt or fresh water. 2. Two major red cell isoenzymes were found in flounder red cells after electrophoresis. These patterns were identical in all fish studied, whether from salt Or fresh water environments. Thus no inherited or adaptive changes were observed. 3. Both flounder red cell carbonic anhydrase isoenzymes split a range of ester substrates. Activity was abolished with acetazolami.'de.
INTRODUCTION The levels of carbonic anhydrase (E.C. 4.2.1.1) isoenzymes in red cells and gills of the euryhaline fish Anguilla anouilla change markedly on adaptation to fresh or sea water (Girard & Istin 1975). These authors found a pronounced shift of isoenzyme pattern and a higher isoelectric point, on adaptation to sea water. The overall enzyme activity increased. In contrast to the above observation, Mashiter & Morgan (1975) found no significant difference in flounder (Platichthys flesus L.) gill or intestinal earbonie anhydras¢ levels after fresh water or sea water adaptation. These later workers also reported an absence of carbonic anhydrase activity, measured using p-nilxophenyl acetate as substrate, from the red cells of flounder. T h e present paper describes the detection of 2 carbonic anhydrase isoenzymes in flounder red ceils. The carbonic anhydrase was examined both qualitatively and quantitatively before and after adaptation to a sea water environment. MATERIALS AND METHODS
Fish Fifty Platichthys flesus L. were caught in fresh water 27.5 km upstream from the mouth of the river Tamar, Devon. The fresh water temperature was 12.7°C. This region of the estuary is fresh water at all states of the tide and flounders in this fresh water zone remain there until washed downstream by a spate (Dando, unpublished studies). No heavy rainfall had occurred for some months prior to the capture of the fish and it is therefore assumed that the flounders were well adapted to fresh water before capture. Blood samples were taken and the fish were transferred to sea water in the laboratory. The sea water temperature ranged from 11.0 to 16.2°C and the salinity from 33.15 to 34.65% during the experiment. A second blood sample was taken between 3 and 6 weeks after transfer to salt water.
Blood samples Samples were drawn from the renal portal vein into 3.8% (W/V) sodium citrate 40% (V/V) glycerol. The cells were washed twice in 0.1 M Triethanolamine, pH 7.0 40% glycerol. 399
The haemolysates were prepared by sonication of blood samples, after addition of an equal vol of distilled water. Haonolysates were spun at 2000g for 20 rain before electrophoresis.
Electrophoretic techniques Electrophoresis was carried out at room temperature on Cellogel strips using a 0.12 M Tris-0.05 M EDTA-0.015 M borate buffer (pH 9.1) for l h r at 400V as previously described (Carter et al., 1973). The gels were stained for protein with nigrosin, and bromothymol blue was used to locate the zone of CO2 hydrase activity (Tashian, 1969). Staining of gels with fluorogenic substrates (umbelliferone acetate and fluorescein diacetate) was carried out as described by Hopkinson et al. (1974). Esterase activity after electrophoresis using p-nitrophenyl acetate as substrate was carded out by making a 1% agar overlay of 3mg p-nitrophenyl acetate in 10ml 0.02 M phosphate buffer, pH 6.5. Esterase zones appeared as yellow bands of p-nitrophenol at the gel agar interface.
Enzyme assays CO2 hydrase activity was measured by the bromothymol blue indicator assay, as originally described by Wilbur & Anderson (1948). The enzymic activity was related to the haemoglobin concentration in the lysates. RESULTS
Electrophoresis Haemolysates from flounders adapted to fresh and to salt water (Fig. 1) showed identical zymogram patterns when the CO2-bromothymol blue stain was used. Two zones of activity were found, one just cathodal to the haemoglobin, and the other anodal to the haemoglobin. The anodal isoenzyme stained dearly with nigrosin. Both carbonic anhydrase zones reacted with the fluorogenic substrates fluorescein diacetate, and umbelliferone acetate; as well as with p-nitrophenyl acetate, as revealed by the production of bright yellow bands of p-nitrophenol. No individual variation was found among the 50 fish tested. Similar zymograms were obtained using blood samples taken from flounders c a p ~ r e d in the lower part of the Tamar estuary (salinity > 25%).
400
N. CARTER, JENNIFERAUTONAND P. DANDO
Carbonic anhydrase " ~ ' ~
memoglobin
Carbonic anhydrase
.ORIGIN
(a)
(b)
Fig. 1. Comparison of flesh water and sea water adapted flounder haemolysates after electrophoresis on Cellogel strips for 1 hr at 400V 10mA, at pH 9.1, when identical patterns were observed. Representation of the CO2 hydrase activity zones are shown. If nigrosine is used to locate protein zones the fast CA is clearly defined, but the slow isoenzyme overlaps the haemoglobin.
Enzyme activity Lysates from 9 sea water adapted fish and 9 fresh water adapted fish, selected at random from each group, were assayed under identical conditions for carbonic anhydrase activity. Haemoglobin concentrations were determined spectrophotometrically at 540 nm using Drabkins reagent. The specific activity of carbonic anhydrase related to haemoglobin in the samples is shown in Table 1. The arithmetic mean and standard deviation for both adaptations were calculated, and no significant difference in carbonic anhydrase levels was found. Stability experiments were also carried out. The flounder carbonic anhydrase was found to retain 100% activity at room temperature (24°C) for up to
5~hr.
The flounder carbonic anhydrase activity was inactivated by incubation with 1.6 mM acetazolamide, as found by Mashiter & Morgan (1975).
flounders adapted to both fresh water and sea water environments. This is in contrast to the findings of Mashiter & Morgan (1975), who found no significant levels of this enzyme in flounder red cells. The apparent lack of this enzyme in the red cells found by these workers might be due to the insensitivity of the esterase assay, with p-nitrophenyl acetate as substrate, in comparison with CO2 used in the present studies. However, using an overlay of p-nitrophenyl acetate on flounder lysate, electrophoresed on Cellogel, we found yellow zones of p-nitrophenol which coincided with the activity zones found using the CO2/bromothymol blue locating method. Two isoenzymes of carbonic anhydrase were found in flounder lysates (Fig. 1). The fast moving (anodal) zone was located with the CO2/bromothymol blue method, fluorogenic esterase substrates (fluorescein diacetate and umbelliferone acetate, p-nitrophenyl acetate, and with the protein stain, nigrosine. The slow moving isoenzyme (Fig. 1) gave approximately equal activity with the CO2/bromothymol blue locating system, with both fluorogenic substrates, and p-nitrophenyl acetate. Fluorogenic substrates have been used to distinguish mammalian CAI ("low activity") and CAII ("high activity") isoenzymes (Hopkinson et al., 1974; Carter & Auton, 1975). In most mammalian systems CAI reacted with umbelliferone acetate, but not with fluorescein diacetate, and the reverse situation was found for CAII. In the flounder lysate both carbonic anhydrase isoenzymes hydrolysed the 2 fluorogenic substrates at approximately equivalent rates. When red cell lysates from flounders were tested after adaptation to sea water the carbonic anhydrase was not significantly different in respect of total red cell activity (Table 1) or qualitative isoenzyme pattern (Fig. 1). This similarity is in accord with the finding of Mashiter & Morgan in flounder tissues (although as previously stated these workers found no activity in red cells). In similar adaptive experiments on other species, marked qualitative and quantitative carbonic anhydrase differences have been found (e.g. Anguilla anguilla) and thus the adaptive role of this enzyme in fish is not yet clear.
DISCUSSION
The results indicate that significant levels of carbonic anhydrase are present in the red cells of
Acknowledoements--We are grateful to the South West Water Authority for permission to trawl in the upper reaches of the Tamar estuary.
Table 1. Fresh water Adapted Units/g Hb Units/ml Hb(g/ml) ( × 10 -3) Ft F2 F3 F4 Fs F6 F7 Fs
460 880 580 216 182 222 376 38 F9 418 Mean = 1.12 x
0.23 0.76 0.47 0.18 0.18 0.18 0.58 0.64 0.35 10-3 __+0.379
2.00 1.16 1.24 1.20 1.00 1.24 0.64 0.84 0.76 x 10 -3
Sea water Adapted Units/g Hb Units/ml Hb(g/ml) ( x 10- 3) Sxo $11 $12 S13 $14 $15 Sl6 St7 Sis Mean
272 344 410 294 228 600 236 386 170 = 1.20 x
0.20 0.15 0.38 0.29 0.18 0.72 0.20 0.32 0.26 10-3 _+ 0.498
1.36 2.40 1.08 1.02 1.26 0.84 0.98 1.20 0.66 × 10 -3
Red cell carbonic anhydrase levels in flounders For assistance in catching and sampling the fish we wish to thank the Skipper and crew of R.L. "Gammarus", Mr. M. Liddicoat and Mr. R. Ling.
REFERENCES
CARTER N. D., TAr~IS R. J., TASHIAN R. E. & FERRELL E. (1973) Characterisation of a new variant of human red cell carbonic anhydrase CAlf London (Giu 102-Lys) Biochera. Genet. 10, 399-408. C~a~a~ N. D. & AuavN J. A. (1975) Characterisation of carbonic anhydrases from tissues of the cat. Biochira. biophys..4cta 410, 220-228. GmARt) J. P. & ISTINM. (1975) Isoenzymes de l'anhydrase carbonique d'un poisson euryhalin. Variations en rela-
401
tion avcc L'osmoregulation. Biochim. biophys. Acta 381. 221-232. HOPK1NSON D. A., COPPOCKJ. S., M[IHLEMANNM. F. & EDWARDsY. H. (1974) The detection and differentiation of the products of the human carbonic anhydrase loci, CAI and CAII, using fluorogenic substrates. Ann. Hum. Genet. 38, 155-162. MASHITER K. E. & MORCAN M. R. J. (1975) Carbonic anhydrase levels in the tissues of flounders adapted to sea water and fresh water. Comp. Biochem. Physiol. 52A, 713-717. TASHIAN R. E. (1969) The esterases and carbonic anhydrases of human erythrocytes. In Biochemical Methods in Red Cell Genetics (Edited by YtJNIS J. J.) pp. 307-336. Academic Press, New York. WILBUR K. M. & ANDERSON N. G. (1948) Electrometric and colorimetric determination of carbonic anhydrase. J. biol. Chem. 176, 147-154.
RED CELL CARBONIC ANHYDRASE LEVELS IN FLOUNDERS, P L A T I C H T H Y S FLESUS L., FROM SALT WATER AND FRESH WATER NICHOLAS CARTER,* JENNIFER AUTON* AND PAUL DANDO~" *Department of Biochemistry, London Hospital Medical College, London, E1 2AD and t Marine Biological Research Association, Citadel Hill, Plymouth, Devon, U.K.
(Received 20 February 1976) AMlract--1. Carbonic anhydrase levels in flounder red cells were unchanged by adaptation to salt or fresh water. 2. Two major red cell isoenzymes were found in flounder red cells after electrophoresis. These patterns were identical in all fish studied, whether from salt Or fresh water environments. Thus no inherited or adaptive changes were observed. 3. Both flounder red cell carbonic anhydrase isoenzymes split a range of ester substrates. Activity was abolished with acetazolami.'de.
INTRODUCTION The levels of carbonic anhydrase (E.C. 4.2.1.1) isoenzymes in red cells and gills of the euryhaline fish Anguilla anouilla change markedly on adaptation to fresh or sea water (Girard & Istin 1975). These authors found a pronounced shift of isoenzyme pattern and a higher isoelectric point, on adaptation to sea water. The overall enzyme activity increased. In contrast to the above observation, Mashiter & Morgan (1975) found no significant difference in flounder (Platichthys flesus L.) gill or intestinal earbonie anhydras¢ levels after fresh water or sea water adaptation. These later workers also reported an absence of carbonic anhydrase activity, measured using p-nilxophenyl acetate as substrate, from the red cells of flounder. T h e present paper describes the detection of 2 carbonic anhydrase isoenzymes in flounder red ceils. The carbonic anhydrase was examined both qualitatively and quantitatively before and after adaptation to a sea water environment. MATERIALS AND METHODS
Fish Fifty Platichthys flesus L. were caught in fresh water 27.5 km upstream from the mouth of the river Tamar, Devon. The fresh water temperature was 12.7°C. This region of the estuary is fresh water at all states of the tide and flounders in this fresh water zone remain there until washed downstream by a spate (Dando, unpublished studies). No heavy rainfall had occurred for some months prior to the capture of the fish and it is therefore assumed that the flounders were well adapted to fresh water before capture. Blood samples were taken and the fish were transferred to sea water in the laboratory. The sea water temperature ranged from 11.0 to 16.2°C and the salinity from 33.15 to 34.65% during the experiment. A second blood sample was taken between 3 and 6 weeks after transfer to salt water.
Blood samples Samples were drawn from the renal portal vein into 3.8% (W/V) sodium citrate 40% (V/V) glycerol. The cells were washed twice in 0.1 M Triethanolamine, pH 7.0 40% glycerol. 399
The haemolysates were prepared by sonication of blood samples, after addition of an equal vol of distilled water. Haonolysates were spun at 2000g for 20 rain before electrophoresis.
Electrophoretic techniques Electrophoresis was carried out at room temperature on Cellogel strips using a 0.12 M Tris-0.05 M EDTA-0.015 M borate buffer (pH 9.1) for l h r at 400V as previously described (Carter et al., 1973). The gels were stained for protein with nigrosin, and bromothymol blue was used to locate the zone of CO2 hydrase activity (Tashian, 1969). Staining of gels with fluorogenic substrates (umbelliferone acetate and fluorescein diacetate) was carried out as described by Hopkinson et al. (1974). Esterase activity after electrophoresis using p-nitrophenyl acetate as substrate was carded out by making a 1% agar overlay of 3mg p-nitrophenyl acetate in 10ml 0.02 M phosphate buffer, pH 6.5. Esterase zones appeared as yellow bands of p-nitrophenol at the gel agar interface.
Enzyme assays CO2 hydrase activity was measured by the bromothymol blue indicator assay, as originally described by Wilbur & Anderson (1948). The enzymic activity was related to the haemoglobin concentration in the lysates. RESULTS
Electrophoresis Haemolysates from flounders adapted to fresh and to salt water (Fig. 1) showed identical zymogram patterns when the CO2-bromothymol blue stain was used. Two zones of activity were found, one just cathodal to the haemoglobin, and the other anodal to the haemoglobin. The anodal isoenzyme stained dearly with nigrosin. Both carbonic anhydrase zones reacted with the fluorogenic substrates fluorescein diacetate, and umbelliferone acetate; as well as with p-nitrophenyl acetate, as revealed by the production of bright yellow bands of p-nitrophenol. No individual variation was found among the 50 fish tested. Similar zymograms were obtained using blood samples taken from flounders c a p ~ r e d in the lower part of the Tamar estuary (salinity > 25%).
400
N. CARTER, JENNIFERAUTONAND P. DANDO
Carbonic anhydrase " ~ ' ~
memoglobin
Carbonic anhydrase
.ORIGIN
(a)
(b)
Fig. 1. Comparison of flesh water and sea water adapted flounder haemolysates after electrophoresis on Cellogel strips for 1 hr at 400V 10mA, at pH 9.1, when identical patterns were observed. Representation of the CO2 hydrase activity zones are shown. If nigrosine is used to locate protein zones the fast CA is clearly defined, but the slow isoenzyme overlaps the haemoglobin.
Enzyme activity Lysates from 9 sea water adapted fish and 9 fresh water adapted fish, selected at random from each group, were assayed under identical conditions for carbonic anhydrase activity. Haemoglobin concentrations were determined spectrophotometrically at 540 nm using Drabkins reagent. The specific activity of carbonic anhydrase related to haemoglobin in the samples is shown in Table 1. The arithmetic mean and standard deviation for both adaptations were calculated, and no significant difference in carbonic anhydrase levels was found. Stability experiments were also carried out. The flounder carbonic anhydrase was found to retain 100% activity at room temperature (24°C) for up to
5~hr.
The flounder carbonic anhydrase activity was inactivated by incubation with 1.6 mM acetazolamide, as found by Mashiter & Morgan (1975).
flounders adapted to both fresh water and sea water environments. This is in contrast to the findings of Mashiter & Morgan (1975), who found no significant levels of this enzyme in flounder red cells. The apparent lack of this enzyme in the red cells found by these workers might be due to the insensitivity of the esterase assay, with p-nitrophenyl acetate as substrate, in comparison with CO2 used in the present studies. However, using an overlay of p-nitrophenyl acetate on flounder lysate, electrophoresed on Cellogel, we found yellow zones of p-nitrophenol which coincided with the activity zones found using the CO2/bromothymol blue locating method. Two isoenzymes of carbonic anhydrase were found in flounder lysates (Fig. 1). The fast moving (anodal) zone was located with the CO2/bromothymol blue method, fluorogenic esterase substrates (fluorescein diacetate and umbelliferone acetate, p-nitrophenyl acetate, and with the protein stain, nigrosine. The slow moving isoenzyme (Fig. 1) gave approximately equal activity with the CO2/bromothymol blue locating system, with both fluorogenic substrates, and p-nitrophenyl acetate. Fluorogenic substrates have been used to distinguish mammalian CAI ("low activity") and CAII ("high activity") isoenzymes (Hopkinson et al., 1974; Carter & Auton, 1975). In most mammalian systems CAI reacted with umbelliferone acetate, but not with fluorescein diacetate, and the reverse situation was found for CAII. In the flounder lysate both carbonic anhydrase isoenzymes hydrolysed the 2 fluorogenic substrates at approximately equivalent rates. When red cell lysates from flounders were tested after adaptation to sea water the carbonic anhydrase was not significantly different in respect of total red cell activity (Table 1) or qualitative isoenzyme pattern (Fig. 1). This similarity is in accord with the finding of Mashiter & Morgan in flounder tissues (although as previously stated these workers found no activity in red cells). In similar adaptive experiments on other species, marked qualitative and quantitative carbonic anhydrase differences have been found (e.g. Anguilla anguilla) and thus the adaptive role of this enzyme in fish is not yet clear.
DISCUSSION
The results indicate that significant levels of carbonic anhydrase are present in the red cells of
Acknowledoements--We are grateful to the South West Water Authority for permission to trawl in the upper reaches of the Tamar estuary.
Table 1. Fresh water Adapted Units/g Hb Units/ml Hb(g/ml) ( × 10 -3) Ft F2 F3 F4 Fs F6 F7 Fs
460 880 580 216 182 222 376 38 F9 418 Mean = 1.12 x
0.23 0.76 0.47 0.18 0.18 0.18 0.58 0.64 0.35 10-3 __+0.379
2.00 1.16 1.24 1.20 1.00 1.24 0.64 0.84 0.76 x 10 -3
Sea water Adapted Units/g Hb Units/ml Hb(g/ml) ( x 10- 3) Sxo $11 $12 S13 $14 $15 Sl6 St7 Sis Mean
272 344 410 294 228 600 236 386 170 = 1.20 x
0.20 0.15 0.38 0.29 0.18 0.72 0.20 0.32 0.26 10-3 _+ 0.498
1.36 2.40 1.08 1.02 1.26 0.84 0.98 1.20 0.66 × 10 -3
Red cell carbonic anhydrase levels in flounders For assistance in catching and sampling the fish we wish to thank the Skipper and crew of R.L. "Gammarus", Mr. M. Liddicoat and Mr. R. Ling.
REFERENCES
CARTER N. D., TAr~IS R. J., TASHIAN R. E. & FERRELL E. (1973) Characterisation of a new variant of human red cell carbonic anhydrase CAlf London (Giu 102-Lys) Biochera. Genet. 10, 399-408. C~a~a~ N. D. & AuavN J. A. (1975) Characterisation of carbonic anhydrases from tissues of the cat. Biochira. biophys..4cta 410, 220-228. GmARt) J. P. & ISTINM. (1975) Isoenzymes de l'anhydrase carbonique d'un poisson euryhalin. Variations en rela-
401
tion avcc L'osmoregulation. Biochim. biophys. Acta 381. 221-232. HOPK1NSON D. A., COPPOCKJ. S., M[IHLEMANNM. F. & EDWARDsY. H. (1974) The detection and differentiation of the products of the human carbonic anhydrase loci, CAI and CAII, using fluorogenic substrates. Ann. Hum. Genet. 38, 155-162. MASHITER K. E. & MORCAN M. R. J. (1975) Carbonic anhydrase levels in the tissues of flounders adapted to sea water and fresh water. Comp. Biochem. Physiol. 52A, 713-717. TASHIAN R. E. (1969) The esterases and carbonic anhydrases of human erythrocytes. In Biochemical Methods in Red Cell Genetics (Edited by YtJNIS J. J.) pp. 307-336. Academic Press, New York. WILBUR K. M. & ANDERSON N. G. (1948) Electrometric and colorimetric determination of carbonic anhydrase. J. biol. Chem. 176, 147-154.