Fish Physiology and Biochemistryvol. 6 no. 2 pp 121-127 (1989) Kugler Publications. Amsterdam/Berkeley
Antifreeze proteins in the urine of marine fish Garth L. Fletcher, Madonna J. King, Ming H. Kao and Margaret A. Shears
Marine Laboratory, Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A 1C 5S7 Keywords: kidney, peptides, freeze-resistance, Pseudopleuronectes sp., Hemitripes sp., Macrozoarces sp., Gadus sp.
Abstract
Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6-33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each o f these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.
Introduction
Many marine teleosts possess antifreeze peptides and glycopeptides which protect them from freezing in ice-laden seawater. These proteins are synthesized in the liver and secreted into the blood and most extracellular fluids where they lower the freezing temperature approximately one degree below that set by the colligative properties (DeVries 1982a; Hew and Fletcher 1985; Fletcher et al. 1986; Davies et al. 1988). All of the antifreezes so far studied are relatively small (2.6 to 33 kd) and most of them should be capable of being filtered through the glomerulus into the urine. However a number of studies suggest that this is not the case, for antifreeze peptides have
not been found in the urine (DeVries 1982a). During the course o f a study o f various body fluids of the winter flounder we discovered the antifreeze proteins were present in the urine. This surprising observation prompted us to examine the urine of several marine fish for antifreeze proteins and to characterize the ones found in the urine of winter flounder.
Materials and methods
Winter flounder ( Pseudopleuronectes americanus) and shorthorn sculpin (Myoxocephalus scorpius) were collected by SCUBA divers from inshore areas of Newfoundland. Atlantic cod (Gadus morhua)
122 were caught by commercial cod traps in Conception Bay, Newfoundland. Sea raven (Hemitripterus americanus) and ocean pout (Macrozoarces americanus) were caught in Passamaquoddy Bay, New Brunswick using a Western trawl and subsequently shipped to Newfoundland. All fish were maintained at the Marine Sciences Research Laboratory in large aquaria (2000 1) for periods of time ranging from one day to 6 months, at seasonally ambient seawater temperature and photoperiod (Fletcher 1977). Sea raven, ocean p o u t and shorthorn sculpin were fed capelin once a week. Winter flounder do not eat during the wintermonths (Fletcher and King 1978). All experiments were carried out during the months of March and April. ~4C-polyethylene glycol (PEG) (4 kd) (New England Nuclear, Mon.treal) was used to indicate the functional status of the renal glomeruli (Renfro 1980). The radioactive isotope was administered intravenously (l o70 NaCl solution) as a single dose using plastic syringes equipped with 25 or 26 gauge needles: shorthorn sculpin and ocean pout 1.5 x 10 6 disintegrations/min (DPM); winter flounder and Atlantic cod 2.0 x 106 DPM; sea raven 5 • 106 DPM. Four days following the injections the fish were killed by a blow on the head and samples of blood and bladder urine were collected using 25 gauge needles and 3 ml plastic syringes. The blood samples were heparinized (Na heparin) in Vacutainers (Becton Dickenson) and the red cells separated from the plasma by low speed centrifugation (~4000 x g). One hundred microliter samples of plasma and urine were digested in Protosol (New England Nuclear) and their radioactivity was determined in Liquifluor (New England Nuclear) using a liquid scintillation counter (Packard, MINAXI Tri-Carb 4000 Series). All counts were corrected for quench using appropriate quench correction curves. Plasma and urine antifreeze activities were determined using a Clifton nanolitre osmometer (Clifton Technical Physics, New York) (Kao et al. 1986). In this method the growth and shrinkage o f ice crystals are observed under a microscope at controlled temperature conditions. The temperature at which the
ice crystal grows is considered the freezing temperature and the temperature at which it melts is the melting temperature. The difference between the freezing and melting temperature has been defined as thermal hysteresis and is a direct measure of antifreeze activity. Antifreeze activity (thermal hysteresis) was converted to antifreeze polypeptide concentrations using the published activity curves for each species: Atlantic cod, Fletcher et al. (1987); all others, Kao el aL (1986). Antifreeze polypeptides from the winter flounder were purified by gel filtration followed by high performance liquid chromatography ( H P L C ) (Fourney et aL 1984a). Urine samples from l0 winter flounder were pooled (5 ml), lyophilized and reconstituted in 1 ml of water. This 1 ml sample was applied to a Sephadex G75 column (0.9 x 60 cm) in 0.1 M NH4HCO 3 and 0.75 ml fractions collected. Plasma samples (2 ml) were applied directly to Sephadex G75 (1.5 x 80 cm) and the antifreeze peptides purified in the same manner as the urine. The antifreeze activity of each fraction was monitored using a Clifton osmometer. Fractions containing antifreeze were pooled, lyophilized and rechromatographed on the same column. The active fractions were again pooled and lyophilized. Aliquots of the Sephadex G-75 purified antifreeze peptides were then dissolved in 5~ formic acid and further fractionated by H P L C using an Altex Ultrasphere ODS reverse phase column (Beckman Instruments) in 0.02 M triethylamine phosphate buffer, pH 3.0 with an acetonitrile gradient (Fourney et al. 1984a). The eluant was monitored at 230 nm. Collected peaks were desalted on Sephadex G-25 and tested for antifreeze activity using a Clifton nanoliter osmometer. Individual H P L C purified peaks (designated urine #'s 10 and 11 and plasma #'s 6 and 8) as well as samples of urine 10 plus plasma 6 and urine I l plus plasma 8, combined in equal proportions, were reapplied to the reverse phase column. Amino acid analysis was conducted on H P L C purified antifreeze peptides from urine. Dried protein samples were hydrolyzed for 24 h at 110~ in 6N HC1. The hydrolysates were analyzed on a Beckman 121 amino acid analyzer.
123 Table 1. P l a s m a and urine concentrations of 14C-PEG and antifreeze peptides
Body Wt. (g)
Urine vol. (ml)
Plasma TH (~
AFP (mg/ml) 11.2 1.77 (11)
Urine
U / P ratio
14C-PEG (DPM)
TH (~
AFP (mg/ml)
t4C-PEG (DPM)
AFP
t4C-PEG
27100 ___1830 (4)
0.422 _+0.081 (11)
7.1 _+ 1.9 (11)
185000 +-44500 (4)
0.70 +-0.017 (11)
7.65 +- 1.05 (4)
Winter flounder
316 +-29.4 (5)
0.56 +_0.14 (5)
0.651 0.044 (11)
Sea raven
1500 +96.0 (3)
0.97 +_0.18 (3)
0.210 +_0.043 (3)
2.9 +0.7 (3)
8840 _ 1650 (3)
0.146 +-0.025 (3)
1.83 -+0.366 (3)
5170 + 1450 (3)
0.84 -+0.24 (3)
0.67 +- 0.24 (3)
Ocean pout
125 +_25.9 (5)
0.37 -+0.14 (5)
0.171 +_0.022 (5)
1.69 _+ 0.276 (5)
34700 _+5440 (5)
0.058 +_0.048 (5)
0.61 +_0.5 (5)
113000 _+27900 (5)
0.32 +_0.23 (5)
3.94 + 1.7 (5)
Cod
425 +- 74.6 (5)
1.1 _+0.49 (5)
0.375 +-0.027 (5)
6.74 _+ 0.66 (5)
12100 _+2330 (5)
0.111 +-0.038 (5)
1.48 _+0.58 (5)
421000 +- 36600 (5)
0.213 _+0.076 (5)
41.0 +_ 10.2 (5)
Shorthorn sculpin
606 _+64.0 (6)
1.72 +_ 1.18 (6)
0.71 _+0.034 (6)
10.4 +_ 0.98 (6)
6578 +_781 (6)
0
0
0
(6)
(6)
(6)
-
-
U / P ratio is the urine to plasma ratio; all values are m e a n s + one SEM (n); 14C-PEG = 14C-polyethylene glycol; A F P = antifreeze peptides; T H = thermal hysteresis; water temperatures of the above experiments were as follows: winter flounder 0.0~ sea raven -0.5~ ocean pout 2.0~ cod 0.5~ shorthorn sculpin - 0 . 5 ~
Results
Antifreeze peptides and 14C-PEG were present in the urine of all fish studied except the shorthorn sculpin (Table 1). Mean antifreeze concentrations in the urine ranged from approximately 2107o of plasma values in cod to 84~ in the sea raven. Urine to plasma ratios of 14C-PEG indicated that cod reabsorbed considerable amounts of water from the urine following glomerular filtration ( U / P ratio = 41.0). Winter flounder and ocean pout reabsorbed lesser amounts ( U / P ratio 7.65 and 3.94 respectively) while there appeared to be a net secretion of water in the sea raven urine ( U / P ratio 0.67). These results on water reabsorption must be considered preliminary since we do not know to what degree a steady state was established between the entry and exit of 14C-PEG from the urinary bladder. Sephadex G75 chromatography of winter flounder urine revealed a single peak of macromolecular antifreeze o f approximately 10 kd (Fig. 1). The elu-
tion position of this peak was identical to that of antifreezes isolated from flounder plasma. Analysis of the G75 purified antifreeze peptides on reverse phase H P L C indicated the presence of at least eleven protein peaks in the urine and eight in the plasma (Fig. 2). The H P L C profile o f the plasma was identical to that reported by Fourney et al. (1984a, 1984b). Plasma componefits ! and 2 are devoid of antifreeze activity, while components 6 and 8 are the major antifreeze species. Of the eleven protein peaks found in the urine only two, components I0 and l 1, possessed antifreeze activity. The elution positions of these two components corresponded to those of plasma components 6 and 8 (Fig. 2). Plasma components 6 and 8 and urine components 10 and 11 were eluted from the H P L C column and reapplied to the column as single components (Fig. 3). The elution times for urine components l0 and 11 were identical to plasma components 6 and 8 respectively. The combination of plasma component 6 and urine component I0 resulted in a single H P L C peak indicating that their elution times were
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Antifreeze proteins in the urine of marine fish Garth L. Fletcher, Madonna J. King, Ming H. Kao and Margaret A. Shears
Marine Laboratory, Ocean Sciences Centre, Memorial University of Newfoundland, St. John's, Newfoundland, Canada, A 1C 5S7 Keywords: kidney, peptides, freeze-resistance, Pseudopleuronectes sp., Hemitripes sp., Macrozoarces sp., Gadus sp.
Abstract
Several species of marine teleosts have evolved blood plasma antifreeze polypeptides which enable them to survive in ice-laden seawater. Four distinct antifreeze protein classes differing in carbohydrate content, amino acid composition, protein sequence and secondary structure are currently known. Although all of these antifreezes are relatively small (2.6-33 kd) it was generally thought that they were excluded from the urine by a variety of glomerular mechanisms. In the present study antifreeze polypeptides were found in the bladder urine of winter flounder (Pseudopleuronectes americanus), sea raven (Hemitripterus americanus), ocean pout (Macrozoarces americanus) and Atlantic cod (Gadus morhua). Since the plasma of each o f these fish contains a different antifreeze class it would appear that all four classes of antifreeze can enter the urine. The major antifreeze components in the urine of winter flounder were found to be identical to the major plasma components in terms of high performance liquid chromatography retention times and amino acid composition. It is concluded that plasma antifreeze peptides need not be chemically modified before they can enter the urine.
Introduction
Many marine teleosts possess antifreeze peptides and glycopeptides which protect them from freezing in ice-laden seawater. These proteins are synthesized in the liver and secreted into the blood and most extracellular fluids where they lower the freezing temperature approximately one degree below that set by the colligative properties (DeVries 1982a; Hew and Fletcher 1985; Fletcher et al. 1986; Davies et al. 1988). All of the antifreezes so far studied are relatively small (2.6 to 33 kd) and most of them should be capable of being filtered through the glomerulus into the urine. However a number of studies suggest that this is not the case, for antifreeze peptides have
not been found in the urine (DeVries 1982a). During the course o f a study o f various body fluids of the winter flounder we discovered the antifreeze proteins were present in the urine. This surprising observation prompted us to examine the urine of several marine fish for antifreeze proteins and to characterize the ones found in the urine of winter flounder.
Materials and methods
Winter flounder ( Pseudopleuronectes americanus) and shorthorn sculpin (Myoxocephalus scorpius) were collected by SCUBA divers from inshore areas of Newfoundland. Atlantic cod (Gadus morhua)
122 were caught by commercial cod traps in Conception Bay, Newfoundland. Sea raven (Hemitripterus americanus) and ocean pout (Macrozoarces americanus) were caught in Passamaquoddy Bay, New Brunswick using a Western trawl and subsequently shipped to Newfoundland. All fish were maintained at the Marine Sciences Research Laboratory in large aquaria (2000 1) for periods of time ranging from one day to 6 months, at seasonally ambient seawater temperature and photoperiod (Fletcher 1977). Sea raven, ocean p o u t and shorthorn sculpin were fed capelin once a week. Winter flounder do not eat during the wintermonths (Fletcher and King 1978). All experiments were carried out during the months of March and April. ~4C-polyethylene glycol (PEG) (4 kd) (New England Nuclear, Mon.treal) was used to indicate the functional status of the renal glomeruli (Renfro 1980). The radioactive isotope was administered intravenously (l o70 NaCl solution) as a single dose using plastic syringes equipped with 25 or 26 gauge needles: shorthorn sculpin and ocean pout 1.5 x 10 6 disintegrations/min (DPM); winter flounder and Atlantic cod 2.0 x 106 DPM; sea raven 5 • 106 DPM. Four days following the injections the fish were killed by a blow on the head and samples of blood and bladder urine were collected using 25 gauge needles and 3 ml plastic syringes. The blood samples were heparinized (Na heparin) in Vacutainers (Becton Dickenson) and the red cells separated from the plasma by low speed centrifugation (~4000 x g). One hundred microliter samples of plasma and urine were digested in Protosol (New England Nuclear) and their radioactivity was determined in Liquifluor (New England Nuclear) using a liquid scintillation counter (Packard, MINAXI Tri-Carb 4000 Series). All counts were corrected for quench using appropriate quench correction curves. Plasma and urine antifreeze activities were determined using a Clifton nanolitre osmometer (Clifton Technical Physics, New York) (Kao et al. 1986). In this method the growth and shrinkage o f ice crystals are observed under a microscope at controlled temperature conditions. The temperature at which the
ice crystal grows is considered the freezing temperature and the temperature at which it melts is the melting temperature. The difference between the freezing and melting temperature has been defined as thermal hysteresis and is a direct measure of antifreeze activity. Antifreeze activity (thermal hysteresis) was converted to antifreeze polypeptide concentrations using the published activity curves for each species: Atlantic cod, Fletcher et al. (1987); all others, Kao el aL (1986). Antifreeze polypeptides from the winter flounder were purified by gel filtration followed by high performance liquid chromatography ( H P L C ) (Fourney et aL 1984a). Urine samples from l0 winter flounder were pooled (5 ml), lyophilized and reconstituted in 1 ml of water. This 1 ml sample was applied to a Sephadex G75 column (0.9 x 60 cm) in 0.1 M NH4HCO 3 and 0.75 ml fractions collected. Plasma samples (2 ml) were applied directly to Sephadex G75 (1.5 x 80 cm) and the antifreeze peptides purified in the same manner as the urine. The antifreeze activity of each fraction was monitored using a Clifton osmometer. Fractions containing antifreeze were pooled, lyophilized and rechromatographed on the same column. The active fractions were again pooled and lyophilized. Aliquots of the Sephadex G-75 purified antifreeze peptides were then dissolved in 5~ formic acid and further fractionated by H P L C using an Altex Ultrasphere ODS reverse phase column (Beckman Instruments) in 0.02 M triethylamine phosphate buffer, pH 3.0 with an acetonitrile gradient (Fourney et al. 1984a). The eluant was monitored at 230 nm. Collected peaks were desalted on Sephadex G-25 and tested for antifreeze activity using a Clifton nanoliter osmometer. Individual H P L C purified peaks (designated urine #'s 10 and 11 and plasma #'s 6 and 8) as well as samples of urine 10 plus plasma 6 and urine I l plus plasma 8, combined in equal proportions, were reapplied to the reverse phase column. Amino acid analysis was conducted on H P L C purified antifreeze peptides from urine. Dried protein samples were hydrolyzed for 24 h at 110~ in 6N HC1. The hydrolysates were analyzed on a Beckman 121 amino acid analyzer.
123 Table 1. P l a s m a and urine concentrations of 14C-PEG and antifreeze peptides
Body Wt. (g)
Urine vol. (ml)
Plasma TH (~
AFP (mg/ml) 11.2 1.77 (11)
Urine
U / P ratio
14C-PEG (DPM)
TH (~
AFP (mg/ml)
t4C-PEG (DPM)
AFP
t4C-PEG
27100 ___1830 (4)
0.422 _+0.081 (11)
7.1 _+ 1.9 (11)
185000 +-44500 (4)
0.70 +-0.017 (11)
7.65 +- 1.05 (4)
Winter flounder
316 +-29.4 (5)
0.56 +_0.14 (5)
0.651 0.044 (11)
Sea raven
1500 +96.0 (3)
0.97 +_0.18 (3)
0.210 +_0.043 (3)
2.9 +0.7 (3)
8840 _ 1650 (3)
0.146 +-0.025 (3)
1.83 -+0.366 (3)
5170 + 1450 (3)
0.84 -+0.24 (3)
0.67 +- 0.24 (3)
Ocean pout
125 +_25.9 (5)
0.37 -+0.14 (5)
0.171 +_0.022 (5)
1.69 _+ 0.276 (5)
34700 _+5440 (5)
0.058 +_0.048 (5)
0.61 +_0.5 (5)
113000 _+27900 (5)
0.32 +_0.23 (5)
3.94 + 1.7 (5)
Cod
425 +- 74.6 (5)
1.1 _+0.49 (5)
0.375 +-0.027 (5)
6.74 _+ 0.66 (5)
12100 _+2330 (5)
0.111 +-0.038 (5)
1.48 _+0.58 (5)
421000 +- 36600 (5)
0.213 _+0.076 (5)
41.0 +_ 10.2 (5)
Shorthorn sculpin
606 _+64.0 (6)
1.72 +_ 1.18 (6)
0.71 _+0.034 (6)
10.4 +_ 0.98 (6)
6578 +_781 (6)
0
0
0
(6)
(6)
(6)
-
-
U / P ratio is the urine to plasma ratio; all values are m e a n s + one SEM (n); 14C-PEG = 14C-polyethylene glycol; A F P = antifreeze peptides; T H = thermal hysteresis; water temperatures of the above experiments were as follows: winter flounder 0.0~ sea raven -0.5~ ocean pout 2.0~ cod 0.5~ shorthorn sculpin - 0 . 5 ~
Results
Antifreeze peptides and 14C-PEG were present in the urine of all fish studied except the shorthorn sculpin (Table 1). Mean antifreeze concentrations in the urine ranged from approximately 2107o of plasma values in cod to 84~ in the sea raven. Urine to plasma ratios of 14C-PEG indicated that cod reabsorbed considerable amounts of water from the urine following glomerular filtration ( U / P ratio = 41.0). Winter flounder and ocean pout reabsorbed lesser amounts ( U / P ratio 7.65 and 3.94 respectively) while there appeared to be a net secretion of water in the sea raven urine ( U / P ratio 0.67). These results on water reabsorption must be considered preliminary since we do not know to what degree a steady state was established between the entry and exit of 14C-PEG from the urinary bladder. Sephadex G75 chromatography of winter flounder urine revealed a single peak of macromolecular antifreeze o f approximately 10 kd (Fig. 1). The elu-
tion position of this peak was identical to that of antifreezes isolated from flounder plasma. Analysis of the G75 purified antifreeze peptides on reverse phase H P L C indicated the presence of at least eleven protein peaks in the urine and eight in the plasma (Fig. 2). The H P L C profile o f the plasma was identical to that reported by Fourney et al. (1984a, 1984b). Plasma componefits ! and 2 are devoid of antifreeze activity, while components 6 and 8 are the major antifreeze species. Of the eleven protein peaks found in the urine only two, components I0 and l 1, possessed antifreeze activity. The elution positions of these two components corresponded to those of plasma components 6 and 8 (Fig. 2). Plasma components 6 and 8 and urine components 10 and 11 were eluted from the H P L C column and reapplied to the column as single components (Fig. 3). The elution times for urine components l0 and 11 were identical to plasma components 6 and 8 respectively. The combination of plasma component 6 and urine component I0 resulted in a single H P L C peak indicating that their elution times were
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