J. exp. Biol. 104, 149-162 (1983)
in Great Britain © The Company of Biologists Limited 1983
149
THE ROLE OF LOW MOLECULAR WEIGHT
ANTIFREEZE GLYCOPEPTIDES IN THE BILE AND
INTESTINAL FLUID OF ANTARCTIC FISH
S. M. O'GRADY*, J. C. ELLORYf AND A. L. D E V R I E S *
• Department of Physiology and Biophysics, 524 Burrill Hall, University
of Illinois, Urbana, Illinois 61801, U.SA. and
f Physiological Laboratory, University of Cambridge, Cambridge, U.K.
CB2 3EG
BY
{Received 20 August 1982—Accepted 19 January 1983)
SUMMARY
The role that low molecular weight antifreeze glycopeptides play in the
physiology of polar fishes has been an open question. In this study, we
demonstrate that antifreeze glycopeptides are present in the bile as well as
the intestinal fluid of antarctic fishes. Isolation of antifreeze glycopeptides
from these fluids by DEAE ion exchange chromatography followed by
polyacrylamide gel electrophoresis revealed the presence of only low
molecular weight glycopeptides (6, 7 and 8). Removal of the gall bladder
with subsequent occlusion of the common bile duct eliminated the transport
of antifreezes into the intestine. This suggests that antifreeze glycopeptides
enter the intestinal lumen by biliary secretion. Measurements of reabsorption, both in vivo and in vitro, indicate that antifreeze glycopeptides are not
reabsorbed as intact molecules. Our results suggest that these glycopeptides
are excreted. We conclude that low molecular weight antifreeze glycopeptides are necessary to prevent the intestinal fluid from freezing and provide
the first clear evidence that low molecular weight antifreeze glycopeptides
have a specific biological function in polar fishes.
INTRODUCTION
Most antarctic fishes synthesize a group of eight glycopeptides that possess unique
antifreeze properties (DeVries & Wohlschlag, 1969; DeVries & Lin, 1977; DeVries,
1980). The larger glycopeptides (1-5) have the greatest antifreeze activity and range
in molecular weight between 10500 and 33700Da (DeVries & Lin, 1977). They
contain two amino acids, alanine and threonine, which are arranged in a repeating
sequence [ala-ala-thr]n. A disaccharide [galactose/3 (1-3) N-acetylgalactosamine] is
attached to each threonine (Sheir, Lin & DeVries, 1975). The smaller glycopeptides,
6, 7 and 8, differ from the larger ones in that proline periodically replaces alanine at
positions 7, 10, 13 and 16. The smaller glycopeptides range in molecular weight from
2600 to 8000 Da and exhibit only one-third of the antifreeze activity of glycopeptides
(1-5).
^ B e y words: Antifreeze, glycoprotein, teleost.
150
S. M. O'GRADY, J. C. ELLORY AND A. L.
DEVRIES
The lowering of the freezing point by these glycopeptides is 100 to 200 times g r e ^
than expected on the basis of colligative relationships. Non-colligative lowering of the
freezing point, without effect on the melting point, is thought to occur through an
absorption-inhibition mechanism where antifreeze molecules bind to the surface of ice
crystals and inhibit their growth (Raymond & DeVries, 1977). This non-colligative
freezing point depression has been referred to as 'antifreeze activity'.
In an earlier study (O'Grady, Ellory & DeVries, 1982), we addressed the problem
of freezing susceptibility of intestinal fluid in polar fishes. Our measurements of
osmolality showed that intestinal fluid and serum of antarctic fishes are nearly isosmotic and significantly hyposmotic to sea water. Since all of these fishes are in constant contact with ice-laden sea water, the intestinal fluid should freeze because ion
concentrations are not high enough to depress the freezing point below environmental
temperature (—2°C) and ingestion of ice precludes the possibility of supercooling.
Freezing and melting point data, together with immunoprecipitation and
polyacrylamide gel electrophoresis results, indicated that antifreeze glycopeptides are
present in the intestinal fluid in high concentration and explained why this fluid does
not freeze. In this study we present a further characterization of these glycopeptides
in the intestinal fluid and demonstrate that antifreeze is also present within the bile.
We also consider the means by which antifreeze glycopeptides enter the intestine and
whether they are excreted or reabsorbed.
MATERIALS AND METHODS
Animals
Specimens of Pagothenia borchgrevinki (Boulenger) and Dissostichus mawsoni
(Norman) were collected by hook and line at McMurdo Sound, Antarctica (77° 54' S
- 166°40' E). These animals were kept in large fibreglass holding tanks in a circulating seawater system. Animals were fed chopped pieces of fish once a week. Tank
temperatures were held at — 1 • 5 °C ± 1 °C and salinity was constant at 35 %c
Blood, bile and intestinal fluid sampling
Blood samples (l 0 ml) were taken from the caudal vein through either an indwelling cannula (D. mawsoni) or 30 gauge needle (P. borchgrevinki). Samples were
allowed to clot for 1 h before centrifugation. After centrifugation at 5000 £ the serum
was removed and frozen for later analysis. Bile samples from D. mawsoni were taken
by removing the gall bladder and emptying the contents into a graduated cylinder.
The sample was then placed in a polyethylene bottle and stored at — 20 °C. Bile
samples from P. borchgrevinki were taken by withdrawing bile from the gall bladder
with a calibrated 1-0 ml syringe. The samples were frozen and stored at — 20 °C.
Intestinal fluid samples were taken by clamping the intestine at the pyloric junction
and anus, then removing the intestine from the body cavity and allowing the contents
to drain into a graduated cylinder. After the volume had been recorded, the sample
was centrifuged and stored in a polyethylene bottle at — 20 °C. In some experiments
where chronic sampling of intestinal fluid from live animals was required, a short
length of polyethylene tubing was inserted into the rectum and the fluid drawn
-
Antifreeze glycopeptides in antarctic
fish
151
a syringe. No evidence of blood contamination was observed in either bile or
intestinal fluid samples.
Osmolcdity and ion analysis
Determinations of major ions and osmolalities were made of serum, bile and intestinal fluid from P. borchgrevinki and D. mawsoni. Osmolalities were measured using
a Wescor vapour pressure osmometer. Sodium and potassium concentrations were
determined by flame photometry using a Corning 455 model flame photometer with
internal lithium standardization. Chloride concentrations were measured with a
Buchler-Cotlove chloridometer.
Freezing and melting point determinations
Freezing and melting points were determined using a cryoscope following the
method of Duman & DeVries (1975). Samples of serum bile or intestinal fluid were
placed in 10/zl capillary tubes and sealed with mineral oil. A small seed crystal was
formed in the sample using a spray refrigerant (Cryokwik) and the sample immersed
in a temperature-controlled viewing chamber ( ± 0-01 °C). Melting points were determined by raising the temperature 0-01 °C every 2min until the seed crystal melted.
This temperature was taken as the melting point of the sample. Freezing points were
determined by lowering the temperature 0-01 °C every 5 min until growth of the seed
crystal was observed. This temperature was taken as the freezing point of the sample.
Solutions containing antifreeze peptides or glycopeptides show a substantial difference between melting point and freezing point by this method.
Purification of antifreeze glycopeptides from bile and intestinal fluid
Antifreeze glycopeptides were isolated from the bile of D. mawsoni and the intestinal fluid of P. borchgrevinki following the procedure of DeVries, Komatsu & Feeney
(1970). Bile (70 ml) and intestinal fluid (12 ml) were dialysed (Spectrapor-3 dialysis
tubing, MW cutoff 3000) against distilled water for 48 h at 4°C. The bile sample was
dialysed a second time (Spectrapor-3) against 2-5 mM-Tris buffer (pH 9-2) at 4°C for
24 h to remove more of the bile salts. Both bile and intestinal fluid samples were
chromatographed on DEAE 22 cellulose columns equilibrated with 2-5 mM-Tris,
pH 9-2. The appearance of antifreeze glycopeptides in the eluent was determined by
monitoring the absorbance at 230 nm with a Beckman double beam
spectrophotometer. Each fraction was dialysed against distilled water to remove Tris
and was then lyophilized. Lyophilized samples were stored at — 20 °C until further
analyses were performed.
*
Amino acid analysis
An amino acid analysis was performed on both bile and intestinal fluid fractions.
One milligram of each fraction was hydrolysed in 6N-HC1 at 110°C for 22 h under a
nitrogen atmosphere according to the procedure of Moore & Stein (1963). The
drolysates were analysed on a Beckman 110 single column automatic amino acid
Myser.
152
S. M. O'GRADY, J. C. ELLORY AND A. L. DEVRIES
Polyacrylamide gel electrophoresis
Polyacrylamide gel electrophoresis was used to determine the specific antifreeze
glycopeptides present in the fractions obtained by ion exchange chromatography.
Both lyophilized fractions were resuspended in 20 fA of borate buffer (0-3 M H3BO3,
pH8-6) at a concentration of 30mgml~1. Fluorescamine (5 jul at 4mgml~ 1 acetone)
was added to each 20^1 sample. The fluorescently labelled glycopeptides were loaded
onto a 10% Bis-polyacrylamide, non-denaturing gel and run at 15 Vcm~' for a period
of 2 h. The gel was analysed under ultraviolet light. The distance that each glycopeptide migrated from the origin was measured and compared with antifreeze glycopeptide standards from D. mawsoni serum.
Localization of antifreeze glycopeptides within the intestine
Two specimens of D. mawsoni were anaesthetized with MS 222. A longitudinal
incision was made along the ventral midline from the pectoral fins to the cloaca to
expose the intestine. Intestinal fluid samples were withdrawn at various points along
the length of the intestine (specifically 10, 20, 35 and 55 cm anterior to the cloaca and
2cm posterior to the pyloric caeca) with a 5 ml syringe (23 gauge needle). In one
animal, stomach fluid was also collected. The samples were centrifuged at 5000^ to
remove debris. Afterwards, osmolality and freezing-melting point determinations
were made.
Distribution of high and low molecular weight antifreeze glycopeptides
Caudal vein cannulae (PE 50 tubing) were placed intofivespecimens of D. mawsoni.
In three animals 3H-labelled antifreeze glycopeptides (1-5) were injected via the
cannula into the caudal vein (4 mg at 1-59 X 10^.p.m. mg" 1 ). In the remaining two
animals 3H-labelled glycopeptide 8 was injected into the caudal vein (lOmg at
0-55 X 108c.p.m. mg" 1 ). After an equilibration period of 1 week, samples of serum,
bile and intestinal fluid were taken. Aliquots of 100 /zl were diluted to 1-0 ml with
distilled water and counted in 9 ml Aquasol. The same experiment was performed
with 11 specimens of P. borchgrevinki using glycopeptides 1-5 (1-59 X 108c.p.m.
mg" 1 ). 7 (0" 53 x 108c.p.m. mg"1) and 8 (0-55 X 10^.p.m. mg" 1 ). One milligram
(dissolved in 100 /J! Ringer solution) was injected into the caudal vein. The injected
antifreeze was allowed to equilibrate for 3 days, after which the animals were killed
and samples of serum, bile and intestinal fluid were taken. The samples were counted
on a Beckman LC 100 scintillation counter. Counts present in the 100 ^1 aliquots were
corrected to d.p.m. Total d.p.m. within each fluid was calculated using an extracellular space volume equal to 12-5 % of the body weight and measured volumes
of bile and intestinal fluid. Estimates of percent extracellular space were calculated
from the ratio of 14C-PEG (polyethylene glycol) in the serum and white muscle off.
borchgrevinki. White muscle was chosen since it makes up most of the body mass of
the fish. To verify that counts appearing in the bile and intestinal fluid are associated
with the antifreeze, portions of bile and intestinal fluid samples were treated with
10% TCA (trichoroacetic acid, antifreeze glycopeptides are soluble in 10% TCA
VanVoorhies, Raymond & DeVries, 1978). The samples were centrifuged
^
Antifreeze glycopeptides in antarctic fish
153
the supernatants dialysed against distilled water for 24 h at 4°C. The samples
were lyophilized and resuspended in distilled water to their original volume. Both
samples were reacted with antifreeze glycopeptide antibody for 24 h at 4°C and then
precipitated overnight in 70% ethanol. The samples were centrifuged (5000 g) and
the supernatant discarded. The precipitates were washed and resuspended in 3-5 ml
of distilled water and counted as a gel in 11*5 ml Aquasol.
Time course of glycopeptide 8 incorporation into bile and intestinal fluid
P. borchgrevinki were anaesthetized (MS 222) and injected with O'Olmg of 3H
glycopeptide 8 (0-55 X 108c.p.m. mg"1) into the caudal vein. After 2, 6, 12, 24 and
47 h, animals were killed and samples of bile and intestinal fluid were taken as
previously described. In a separate experiment, gall bladders were removed from five
specimens and the common bile duct tied shut. Animals were allowed to recover for
48 h, and 0-01 mg 3H glycopeptide 8 was then injected into the caudal vein. Samples
of intestinal fluid were taken with a short length of PE 50 tubing attached to a 1 ml
syringe. Both bile and intestinal fluid samples were treated with 10% TCA. The
samples were centrifuged (5000 g) and the supernatants dialysed against distilled
water for 24 h at 4 °C. The supernatants were lyophilized and resuspended in distilled
water to their original volume. Aliquots (100 ^tl) were diluted to 1-0 ml with distilled
water and counted in 9 ml Aquasol.
Antifreeze reabsorption experiments
P. borchgrevinki were anaesthetized and a small incision (1 cm) was made through
the body wall just anterior to the cloaca. The distal portion of the small intestine was
exposed and a ligature was placed around the intestine. Before the intestine was tied
shut, a short length of PE 50 tubing was inserted through the cloaca 5 cm into the small
intestine. 0-2 mg of 3H-labelled glycopeptide 8 was injected into the small intestine.
The PE 50 tubing was withdrawn and the intestine tied shut. Samples of blood were
taken from the caudal vein at 24 h intervals for a period of 5 days. A portion of the
sample (50 /il) was diluted to 10 ml in distilled water and counted in 9 ml Aquasol.
Pooled portions (50/il each fish) from five animals each day were dialysed against
distilled water for 24 h and then lyophilized. Afterwards they were resuspended in
distilled water at the original volume (0-250 ml). A 50/il aliquot was removed, diluted
to lml in distilled water, and counted in 9 ml Aquasol. Animals usually survived
longer than 10 days. No food was given during this time.
In two specimens of D. mawsoni, an incision along the ventral midline from the
pectoral fins to the cloaca was made and the intestine exposed. 3H-labelled glycopeptide 8 (lOmg at 0*55 X 108c.p.m. mg"1 in 5-0ml sea water) was injected (using
a 30 gauge needle and 5 ml syringe) into the lumen of the intestine along the entire
length. The intestine was not tied shut. The body wall was sutured and the animal
returned to its holding tank. Blood samples (1-0 ml) were drawn each day from an
indwelling cannula located in the caudal vein. Serum samples (100/il) were diluted
and counted as described above. Animals usually survived between 10 and 14 days,
extracellular space volumes were estimated on the basis of body weight
%) and the total number of counts present within the blood calculated in both
154
S. M. O'GRADY, J. C. ELLORY AND A. L. DEVRIES
P. borchgrevinki and D. mawsoni. This value was divided by the total number^p
antifreeze counts injected into the gut and expressed as % total counts present within
the serum.
Isolated intestinal sac experiments
Reabsorption of low molecular weight glycopeptide antifreeze was investigated
using isolated everted sacs. Whole intestines from P. borchgrevinki were removed,
everted and tied off at the anus. A cannula was inserted into the duodenum and tied
in place. The entire gut was suspended in nototheniid Ringer (250 mM-Na+, 5 mM-K+,
3 mM-MgSO 4, 2 mM-NaHCCh , 2 miu-NaHPCU, 5 mM-alanine, 5 miw-glucose,
2-5mM-CaCl2) and held at — 1-5°C. In each sac, approximately 400/xl of nototheniid
Ringer was added to the serosal side. To the mucosal side 3H-labelled antifreeze
glycopeptide 8 (l-0mg in 10-0 ml Ringer) was added. Every 2h 20/il of serosal fluid
was removed, diluted and counted. In a separate experiment 5 fiCi of HC-PEG was
added to the serosal side and no antifreeze was added to the mucosal side. Every 2h
20 fj\ of serosal fluid was removed and counted. This was done as a check to see that
the sacs were alive and transporting fluid. After 6h, 1-5 X 104M-ouabain was added
to the serosal side of the sac containing 14C-PEG. At the end of the experiment, the
total number of counts present in the serosal fluid was calculated at each time point
taking into consideration the volume present after sampling.
RESULTS
Ion concentrations, oamolalities, and freezing-melting point analyses of serum,
bile and intestinal fluid from P. borchgrevinki and D. mawsoni are shown in Table 1.
The osmolalities of bile and intestinal fluid in both species were approximately isosmotic to serum. Chloride concentrations of bile were significantly lower (P<0 - 01)
than chlorides in either serum or intestinal fluid. Differences between freezing and
melting points in all three fluids indicated that a substantial amount of antifreeze is
present in each fluid. Bile volumes in£>. mawsoni were quite variable, ranging between 0-14 and 0-36 ml kg"1. Intestinal fluid volumes were also variable and ranged
between 0-80 and 1-7 ml kg"1.
The elution profile for the purification of glycopeptide antifreeze from D. mawsoni
bile is shown in Fig. 1. The elution profile for antifreeze isolated from P. borchgrevinki intestinal fluid is shown in Fig. 2. Each peak was examined for the presence of
antifreeze activity using melting-freezing point analyses. Antifreeze activity was
observed in both fractions. Amino acid analysis (Table 2) of antifreeze glycopeptides
isolated from the bile and intestinal fluid showed the same amino acid composition as
glycopeptides 7 and 8 from D. mawsoni serum.
Polyacrylamide gel results from D. mawsoni bile and P. borchgrevinki intestinal
fluid are shown in Fig. 3. The bile from D. mawsoni contained a small amount of
glycopeptide 6 along with glycopeptides 7 and 8. The intestinal fluid contained
glycopeptides 7 and 8 with a trace amount of a smaller glycopeptide fragment (8+).
This fragment was observed earlier from TCA-treated extracts of intestinal fluid from
D. mawsoni shown in track 8.
4
Dissostichus
mawsoni
9-6± 1-2
221 ±58
711 ± 58
538 ± 42
608 ± 34
5
8
5
8
4
589 + 14
598 ± 21
6
.V
578 ±12
OSM±s.E.
mosmolkg"'
OSM = osmolality; MP = melting point; FP = freezing point; A'= no. of observations.
249 ± IS
149 ±10
6
Intestinal fluid
Pagothenia
borchgrevinki
8-3 ±0-9
67 ± 19
8-4 ±0-6
318 ± 13
4
196 ± 12
88 ±16
9-3 ±0-8
308 ± 18
5
261 ± 8
4-7 ±0-8
278 ± 9
5
Bile
Pagothenia
borchgrevinki
Dissostichus
mawsoni
256 ±10
5 1 ± 0-6
262 ± 11
5
Cr±s.E.
mequivl"1
Serum
Pagothenia
borchgrevinki
Dissostichus
mawsoni
K + ±s.E.
mequivP 1
.V
Species
Na + ±s.E.
mequivl"'
01-47 ±0-54
- 1 04 ± 0 0 8
- 1 08 ± 0 0 3
-1-07 ±0-02
—1-11 ± 004
-1-08 ±0-06
MP + s.E.
-2-56±0-07
-2-54±0-07
-202 ± 0 1 0
-2-25 ± 0 0 8
-2-62 ± 0 0 4
-2-71 ± 0 0 8
FP + s.E.
109
1-50
0-94
118
1-51
1-63
MP-FP
5
7
7
5
.V
l-36±0-32
1-98 ±0-11
0-23 ± 0 0 4
1-20 ± 013
Volume kg '
(ml)
Table 1. Ion concentrations, osmolalities andfreezing-melting point analyses of serum, bile and intestinal fluid from two antarctic
fishes, Pagothenia borchgrevinki and Dissostichus mawsoni
a
to
a-
i
156
S. M. O'GRADY, J. C. ELLORY AND A. L. DEVRIES
20
30
Fraction number
50
Fig. 1. An elution profile for the purification of antifreeze glycopeptides from the bile of D. mawsoni
(O)O.D.230.
10
20
30
Fraction number
40
SO
Fig. 2. An elution profile for the purification of antifreeze glycopeptides from the intestinal fluid of
P. borchgrevinki (A) O.D.ao .
Localization of antifreeze glycopeptides within the intestine
Osmolalities and freezing-melting point differences at various sites along the intestine of D. mawsoni are shown in Fig. 4. Stomach content osmolalities are
approximately the same as sea water (1050mosmolkg~'). A decrease in osmolality
occurs along the intestine towards the cloaca. Differences between melting and freezing points demonstrate that antifreeze is present in the intestinal fluid along the entire
length of the intestine. The amount, however, is highest at the level of the
^^
Antifreeze glycopeptides in antarctic fish
157
2. Amino acid compositions of antifreeze glycopeptides isolated from the serum,
bile and intestinal fluid of antarctic fishes
Alanine
Thrconine
Proline
Galactosamine
D. matvsom scrum
Glycopeptides 7, 8
D. mawsoni bile
Glycopeptides 6, 7, 8
1
0-47
0-14
0-45
1
0-47
0-19
P. borchgrevinki
Intestinal fluid
(Glycopeptides 7, 8, 8+)
0-48
016
0-46
Values expressed as amino acid ratios where alanine (of highest concentration) is given the value of 1.
1 - Antifreeze glycopeptides (1-5)
2 - Antifreeze glycopeptide (6)
3 - Antifreeze glycopeptides (7, 8)
4-Bile
5 - Intestinal fluid (column purification)
6 - Intestinal fluid (after TCA precipitation)
Fig. 3. Polyacrylamide gel showing antifreeze glycopeptides isolated from bile, intestinal fluid and
scrum of D. mawsoni and P. borchgrevinki. Track 1 shows glycopeptide antifreezes 1-5 from the
serum of D. mawsoni; track 2 shows glycopeptide 6 from the serum of D. mawsoni (glycopeptide 6
is a heterogeneous fraction containing four discrete bands); track 3 shows serum glycopeptides 7 and
8 from D. mawsoni; track 4 shows glycopeptides 6, 7 and 8 from the bile of D. matvsoni (glycopeptide
6 was present in trace amounts); track 5 shows glycopeptides 7, 8 and 8+ from the intestinal fluid of
P. borchgrevinki (8+ was present in trace amounts); and track 6 shows glycopeptides 7, 8 and 8+
isolated by TCA precipitation from the intestinal fluid of D. mawsom.
where osmolalities are lowest. In a separate experiment, stomach fluid was collected
from five specimens of P. borchgrevinki. The mean osmolality from five animals was
^ K 5 ± 36-4mosmol kg"1. Thus, stomach fluid is isosmotic to sea water.
EXB 104
158
S. M. O'GRADY, J. C. ELLORY AND A. L. DEVRIES
Pvloric caeca
Stomach
Small intestine
OSM
629
650
2
645
660
3
665
680
4
710
760
5
800
840
FP-MP
1-32
1-26
1-22
131
110
104
0-97
0-92
0-90
0-95
6
992
Fig. 4. A diagrammatic representation of the intestine from D. mavisoni showing the relative
positions at which samples were taken by needle and syringe. The table below shows the values of
osmolality and F P - M P obtained from two specimens of D. mavisoni. OSM = osmolality (mosmol
kg"'); FP—MP = freezing point—melting point.
Table 3. Distribution of high and low molecular weight3H-antifreeze glycopeptides in
the serum, bile and intestinal fluid of Dissostichus mawsoni and Pagothenia borchgrevinki
Serum
(c.p.m. X 10"7)
Dissostichus mawsoni
Bile
(c.p.m. X 10~7)
Intestinal fluid
(c.p.m. X 10"7)
4-3
5-7
10-3
12-8
13 2
0-87
0-37
<0-01 (2500 c.p.m.)
< 0 0 1 (3800 c.p.m.)
<0-01 (4400 c.p.m.)
0-10
008
<0-01 (500 c.p.m.)
< 0 0 1 (1250 c.p.m.)
<0-01 (2000 c.p.m.)
N
Serum
(c.p.m. X 10"5)
Pagothenia borchgrevinki
Bile
(c.p.m. X 10"s)
Intestinal fluid
(c.p.m. X 10"5)
4
4
3
3-47 ±0-31
4-04 ±0-20
7-89 ±0-6
Glycopeptidc
8
1-5
Glycopeptide
7
8
1-5
0-68 ±0-04
0-93 ± 0 - 1 1
<0-01 (1328±56)
0-05 ±0-006
0-02 ±0-001
<0-01 (688 ±40)
Distribution experiments
The distribution of high and low molecular weight glycopeptides from the serum
into bile and intestinal fluid of D. mawsoni and P. borchgrevinki is shown in Table 3.
Counts corresponding to glycopeptide 1-5 in both D. mawsoni and P.
Antifreeze glycopeptides in antarctic
fish
159
) almost completely excluded from the bile and intestinal fluid. However, glycopepIT3es 7 and 8 appear in bile. Glycopeptides 7 and 8 are also present in intestinal fluid.
The appearance of glycopeptide 8 from the serum into the bile and intestinal fluid
of P. borchgrevinki is shown in Fig. 5. The glycopeptide appears more rapidly in bile
than in intestinal fluid. When the common bile duct is tied shut, the appearance of
antifreeze glycopeptide 8 into the small intestine is almost completely eliminated.
These results suggest that the pathway by which low molecular weight glycopeptides
enter the intestine is through the bile and into the intestinal fluid. Although some
secretion from the blood into the intestinal lumen by the gut cannot be completely
ruled out, it appears that it is not the major pathway by which antifreeze enters the
small intestine.
Reabsorption experiments
The results of in vivo reabsorption experiments from P. borchgrevinki and D.
mawsoni are shown in Fig. 6. Reabsorption of labelled antifreeze from the intestine
of two D. mawsoni into the blood was less than 1 % of the total label injected into the
intestine. When the intestine was tied closed as shown with P. borchgrevinki, the
number of counts within the serum increased with time. Over a 5-day period this
increase amounted to approximately 1-5% of the total counts present within the
intestine. After dialysis of the serum, however, nearly all of the counts were
eliminated. This suggests that counts appearing in the serum were not a measure of
intact glycopeptide 8 crossing the intestine into the blood but were probably caused
by the gradual absorption of degradation products of the antifreeze by the intestine.
The question of reabsorption was investigated further by the use of isolated intestinal sacs (Fig. 7). The 14C-PEG dilution experiment demonstrates that the isolated
47
Fig. 5. Accumulation of glycopeptide 8 from serum into bile and intestinal fluid of P. borchgrevinki.
(O) /V = 5; accumulation of glycopeptide 8 into the bile; (A) N = 5; accumulation of glycopeptide
8 into the intestinal fluid; (A) A' = 5, except at 24 and 47 h where A^= 4; accumulation of glycopeptide 8 into the intestinal fluid when the common bile duct was tied shut.
160
S. M. O'GRADY, J. C. ELLORY AND A. L. DEVRIES
2
3
Time (days)
Fig. 6. In vivo reabsorption measurements of 3H-glycopeptide 8 from P. borchgrevinki and D.
mawsom. (O) Reabsorption data for P. borchgrevinki; each value shows the mean±s.E. of five
animals where the distal portion of the small intestine was tied off. (A) Reabsorption data for D.
mawsom; represents the mean of two fish. ( • ) Pooled samples of dialysed serum taken from the five
P. borchgrevinki shown above (O).
- 7
280 -
4
6
10
Time (h)
Fig. 7. Isolated intestinal sac results from P. borchgrevinki. ( • ) Shows the results of one sac experiment where fluid transport was estimated following label dilution of I4 C-PEG. Ouabain ( 0 ) was
added at 6 h and was shown to block fluid transport into the isolated sac. (A) Mean ± s.E.; % uptake
of 3H-labelled glycopeptide 8 by four isolated intestinal sacs incubated in nototheniid Ringer for 10 h
at-15°C.
sacs were viable and could effectively transport fluid. The fact that ouabain effectively
stopped fluid transport indicates that fluid transport is dependent upon sodium pump
activity. Over a period of 10 h essentially no incorporation of label occurred in four
intestines. These results are consistent with our in vivo observations which indicate
that antifreeze is not reabsorbed as an intact glycopeptide.
An tifreeze glycopep tides in an tare tic
fish
161
DISCUSSION
Most marine teleosts are hyposmotic to sea water (Prosser, 1973). This generalization applies to antarctic fishes, although their serum osmolalities are about
ZSOmosmolkg"1 greater than temperate marine species (O'Grady & DeVries, 1982;
Dobbes & DeVries, 1975). As a result of being hyposmotic to sea water, marine fishes
are faced with a continuous osmotic loss of water. To replace osmotic and urinary
water losses, marine fishes drink sea water and absorb water from the gut (Smith,
1930). To facilitate water absorption, sodium and chloride are actively transported
from the intestinal lumen into the extracellular space setting up an osmotic gradient
that pulls water from the gut into the blood (Ramos & Ellory, 1981; House & Green,
1965). Accumulated NaCl is excreted from the blood at the gills. Measurements of
intestinal fluid osmolality along the length of D. mawsoni intestine showed that
intestinal fluid becomes increasingly hyposmotic to sea water as it approaches the
anus. Intestinal fluid at the level of the hind gut is approximately isosmotic to the
blood. Amounts of antifreeze as indicated by differences in freezing and melting
points are also highest in the hind gut, presumably because of the removal of water
along the intestine. Thus, while intestinal contents are most susceptible to freezing
at the hind gut, they are protected from freezing because of high concentrations of
antifreeze glycopeptides.
The manner in which low molecular weight glycopeptides enter the bile is an open
question. Since only low molecular weight glycopeptides are found in the bile, the
simplest explanation is that they are filtered across the tight junctions separating the
lateral spaces from the bile canaliculus. In mammals, serum proteins (at less than
0*2% of their serum concentrations) presumably enter the bile by diffusion across
these tight junctions (Mullock & Hinton, 1982).
Purification data presented in this paper confirm our earlier findings that only low
molecular weight glycopeptides are present in the intestinal fluid. The way in which
antifreeze glycopeptides enter the intestine appears to be by means of biliary
secretion. It is not known whether antifreeze is secreted into the intestine in a slow
continuous manner or if stimulus-induced secretion is involved. In higher
vertebrates, bile is secreted into the intestine as part of the migrating motor complex
during the inter-digestive period. The discharge of bile into the duodenum during the
inter-digestive motor cycle can be as much as one-third of the total bile volume
(Malagelada, 1981). Thus, even during fasting conditions a cyclic pattern of biliary
secretion occurs. Ingestion of water does not significantly affect inter-digestive motor
patterns or biliary secretion (Malagelada, 1981). In antarctic fishes it is conceivable
that the delivery of bile to the duodenum also occurs in a regular cyclic pattern. This
pattern may be subject to modification by drinking since, from a freezing avoidance
viewpoint, ingestion of ice could occur that would lead to freezing injury.
Once antifreeze glycopeptides enter the intestine it appears that little, if any, reabsorption of intact glycopeptide occurs. The fact that a smaller antifreeze fragment,
glycopeptide 8 + , exists in intestinal fluid samples implies that some degradation of
these glycopeptides takes place. It is likely that some of the amino acids and sugars
recovered, but it is evident that much of the intact antifreeze at the hindgut is
eted. The rate of antifreeze excretion is probably dependent upon the dietary
«
162
S. M. O'GRADY, J. C. ELLORY AND A. L. DEVRIES
condition and the rate of digestion of the animal. Antarcticfishessurvive on a diet ^
in lipid. Previous studies on digestion in fishes has shown that the presence of high
lipid in the diet slows down the digestion rate and the rate of gastric emptying
(Kapoor, Smit & Verighina, 1975). The low temperature at which these fishes live
would also depress the rate of digestion, intestinal absorption and excretion. Thus,
given the above considerations, the excretion of antifreeze from the intestine is probably a slow process. The energy lost due to resynthesis of unrecoverable antifreeze in
the intestine may reflect one of the costs of living at temperatures near the freezing
point of sea water.
The authors wish to thank W. VanVoorhies and S. Munsel for their help in collecting and sampling fish at McMurdo Sound, Antarctica. We also thank J. Schrag, J.
Turner and C. O'Grady for reviewing the manuscript and J. Ahlgren for his help in
isolating glycopeptides from the bile and intestinal fluid. This work was supported by
NSF Grant DPP-78-23462 to ALD. JCE thanks The Royal Society for a travel
grant.)
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