Biological antifreeze agents in coldwater fishes

Camp. B~ochnn Physiol. Vol. 73A. Printed m Great Brnnin.

No.

4. pp. 627

to 640.

0.300-9629 X7,170627-l4S03.00 0 cs 19x2 Pcrgamon Press Ltd

1982

BIOLOGICAL ANTIFREEZE AGENTS COLDWATER FISHES ARTHUR Department

of Physiology

L.

DEVRIES

and Biophysics, University IL 61801. USA (Rrceicrd

IN

2 March

of Illinois,

Urbana.

1982)

Abstract-l. Biological antifreezes are present in coldwater fishes and lower their freezing point below the freezing point of seawater (- 1.9 C). without substantially increasing the osmotic content of their body fluids. 2. The antifreeze agents present in the Antarctic notothenioid and northern gadid fishes are glycopeptides composed of alanine. threonine, galactose and N-acetylgalactosamine. In other northern fishes they are peptides which differ substantially in composition between species. but are similar in that two thirds of their residues are alanine and they are rich in the polar residues aspartate. glutamate. threonine and serine. 3. Although the average mol. wt of the antifreezes is between 5000 and 10.000. the! have a very large effect, on the freezing point of water which has been termed an antifreeze effect. The depression of the freezing point occurs via the non-colligative mechanism. adsorption-inhibition. Adsorption tn ice prevents water from Joining the ice lattice by increasing the radius of curvature of the fronts on the growth steps of the crystals thereby increasing their surface free energy. 4. The antifreezes are a permanent feature of the Antarctic fishes. but in many northern tishcs they are present only during the winter and the antifreeze cycle appears to be an endogenous one. 5. Antifreeze concentrations in the blood are between 3 and 4”,,. but they are not present in the urme. In the Antarctic and many northern species. the antifreezes are conserved m the circulation because the kidneys are aglomerular or functionally aglomerular. In the glomerular winter flounder, the acidic peptide antifreeze is conserved in the circulation because it is not tiltrred. Retention of the pcptidc occurs because it is repelled from the negatively charged basement membrane of the capillary wall. Small amounts of low mol. wt antifreeze are lost via the bile and digestive system where their role appears to be prevention of freezing of the dilute intestinal fluid.

INTRODUCTION

temperature of the shallow waters of the polar oceans are near their freezing point and ice-covered for most of the year (Littlepage, 1965). In the high latitudes. where ice cover is abundant, the upper part of the water column is often filled with minute ice crystals and masses of ice platelets called anchor ice, which adhere to the bottom to depths of 30 m (Dayton et al.. 1969). During the winter near-shore waters of many areas of the north-temperate oceans are also covered with ice and the water is near its freezing point of - 1.9”C. These frozen areas are often shallow bays and they are found as far south as Long Island, New York. on the eastern seaboard. while in the Pacitic Ocean freezing waters extend southwards only as far as the Aleutian Islands. In the northeastern Atlantic Ocean, the coast of Norway is ice-free during the winter as far north as the Arctic Circle. Whether or not the coastal waters of the world’s continents freeze appears to be related to the northward flow of major warm currents such as the Gulf Stream and the Japanese Current. The, body fluids of tropical and temperate marine teleosts freeze at temperatures between - 0.5 and - 0.9 C (Black, 1951). Thus fishes inhabiting freezing environments are in danger of freezing unless they elevate their body temperature or increase the osmotic content of their body fluids. In the absence of ice, at these temperatures many fishes can exist in a super-

The

cooled state for their entire lives (Scholander p’t (I/., 1957). In shallow areas. ice is always present when the temperature of seawater is at its freezing point and therefore supercooling is not possible. In almost all of these ice-laden areas of the world’s oceans. a relatively rich fish fauna exists (Andriashev. 1970; Norman, 1938). In fact some fishes utilize the ice as a habitat for feeding and cover. Figure 1 shows the naked dragon fish. G~wnodruco ucuticrps, resting under a mass of anchor ice in 10m of water in McMurdo Sound, Antarctica where the water temperature is - 1.9 C. BEHAVIORAL

AVOIDANCE

OF

FREEZWG

Many organisms leave their summer range to escape the cold. The long horn sculpin, M_ro.uocrpllultrs octodrcemspinosus. leaves the shallow waters of the New England coast in late autumn and spends the winter in waters at + 4 C (Leim & Scott. 1966). Adult specimens of the cunner. Tuutogo/uhru.s ctdsprrus. leave the inshore waters when temperatures begin to decline in the autumn. The juveniles, however, remain in the inshore waters. They overwinter in burrows in the sand in a state of torpor (Olla (‘r ctl.. 1975) and often appear to be supercooled. If such torpid. supercooled specimens are touched with a piece of ice. they quickly freeze and die (Olla B. L.. personal communication). In the fjords of Labrador. bottom temperatures are _ l.KC throughout the year and several species of 127

ARTHI:K L. DEVWFS

628

Fig. I. The naked dragon Bsh. GJWI~~WL.~Iuc~~ti~qw, resting on the hottom nf McMurdo Sound beneath it mass of anchor ice in IO m of water. The ice crystal formations provide ;I safe refuge in u hich this tish can escape its predators.

fish exist there throughout their lives (Scholander cr (I/.. 1957). They are supercooled by approx I’C at their habitat depth of 300m. This small degree of supercooling appears to be sufficiently stable so that freezing does not occur over the lifespan of the fish as long as it does not come into contact with ice. The presence of ice at these depths is unlikely because of the effect of pressure on the freezing point of water. Therefore because of the absence of ice the supercooled fluids of these fishes are not inoculated. The supercooling of the body fluids of fish in the absence of ice is not unusual. This small degree of supercooling therefore must be metastable. The stability of the supercooled state in such fishes would appear to indicate the absence of endogenous nucleators. Supercooling in insects by IO-20 C for months is common and indicates the stability of the supercooled state in the absence of nucleators or ice (Ring, 1980). Habitat selection allows certain fishes to exist in ice-covered oceans in some areas of the Antarctic Ocean. ~otother~i~~ lierrq~i inhabits the ice-covered waters near the Antarctic Circle by remaining in a +2 C layer of water that exists year round at 100-200 m (DeVries. 196X; DeVries & Eastman, 19x21. FREEZING

BEHAVIOR

OF FISHES

most important and often neglected observations in studies of freezing avoidance of fishes is the temperature at which the specimen will freeze in the presence of ice. Since supercooling is common in the absence of ice. seed crystals must always be present to obtain the organismal freezing point. which of course is the meaningful parameter in terms of whether the fish survives or not. This observation has been neglected in many studies (Pearcey. 1961: Smith & Paulson, 1977). In general there is agreement between the organisma1 freezing point of a fish and the temperature at which ice will propagate in its blood or eutraceliulai fluid. For almost all coldwater fishes the blood freezing points are a few tenths of a degree lower than the freezing temperature of the specimen, indicating that freezing is probably initiated in some fluid other than the blood. There is also a correlation between the blood freezing point. the fishes’ freezing tcmperaturc and environmental temperature. Fishes living in the coldest environments such as McMurdo Sound, Antarctica. freeze at lower temperatures and have low blood freezing points, while those living in the warmer sub-Arctic waters have much higher freezing temperatures and freezing points (DeVries. 1980). FREEZING

BEf-IAVIOR OF BODY FLC‘IDS OF POLAR

FISHES

When

fishes freeze and thaw, they do not recover from the effects of it. This is true even if they are only partly frozen (Scholander et (II., 1957). One of the

The freezing point of a solution is defined as the temperature at which the vapor pressure of the solid

Antifreeze

6’9

agents in coldwater fishes

TECHNIQUE FOR FREEZING POINT ~TERH~NATI~S

MINERAL OIL

Fig. 2. Technique used for dctcrmininf freoling point and m.p. of plasma and solutions of pure antifrcerc. The freezing point of the solution is the temperature at ahuh ice propagation occurs at the face of the seed. Crystal growth is always in the form of long spicules Mhosc long axes arc parallel to the c-axis which 1s the non-prcfcrrcd axis of grobyth.

phase (ice) is equal to the vapor pressure over the liquid phase (Pauling. 1953). In practice an estimate of the freezing point of a solution can be obtained by observing the temperature at which a small seed crystal melts as the temperature is slowly raised (0.01 ‘C;min) in a small sample. This by definition is the equilibrium freezing point of the solution. If the temperature is lowered one or two hundredths of a degree before the seed crystal is completely melted, then it can be observed to increase slowly in size. This temperature is also very close to the equilibrium freezing point of the solution. For salt solutions or blood plasma of temperate marine fishes, the freezing point and temperature of melting of the solid phase can be rapidly determined with an accuracy of 0.01 C if the volumes are small. When this freezing point technique is employed for determinations of the blood freezing points of polar fishes. the melting of a seed crystal is near - I-C, a temperature expected on the basis of the number of particles in solution. This m.p. depression is due primarily to NaCl (O’Grady & DeVries, 1982). However. when the temperature is lowered. in the presence of the seed crystal no growth is observed until the tcmperature is lowered to - 2.2 C. In contrast to the hexagonal crystal growth that occurs in salt and other biological solutions, the type that occurs in polar fish blood is rather different. Growth from the seed crystal face is in the form of long spicules which appear much like strands of glass wool (Fig. 2) (DeVries, 1971: Raymond & DeVries. 1977). These spicules rapidly propagate throughout the sample in less than a sec. Their melting temperature is the same as that of the seed crystal. This unusual separation of the m.p. and “freezing point” (temperature of ice propagation) is associated with glycopeptides and peptides that lower the freezing point in a non-colligative manner (DeVries & Wohlschlag. 1969: DeVries. 1971; Duman & DeVries. 1976, Raymond et cd., 1975). Because of their unusual effect on the freezing point these compounds can be described as having antifreeze properties. The presence of these antifreeze compounds in the blood can easily be detected and their concns. estimated by (‘BP ??.l-,

determining the freezing point-m.p. difference of plasma or dialysed plasma (DeVries, 1974). In polar fishes in which the blood freezes at - 7.2 C. 1.2 of the freezing point depression is due to either glycopeptide or peptide antifreeze. Upon dialysis the freezing point of polar fish blood rises to - I.2 C and the m.p. to - 0.02 C. The freezing point and the m.p. of the dialyzed blood of a temperate fish are - O.Ol‘C indicating no antifreeze is present and only salts and other small solutes contribute to its freezing point depression (Fig. 3). BIOLOGICAL

ANTIFREEZES

IK

COLDWATER

FISHES

Two basic types of antifreeze have been isolated from polar and north-temperate fishes which include glycopeptides and peptides. In all of the Antarctic fishes. with one exception, the antifreezes are glycopeptides (DeVries & Lin, 1977a). They have been most thoroughly characterized in the family Nototheniidae. The gfycopeptides are composed of repeating units of gfycotripeptides in which the disaccharide I-D-galactopyranosyl-( 1 --+ 3)-2-acetamido-2-deoxy-rD-gaiactopyranose is linked to the threonine residue of the tripeptide alanyl-threonyl-afanine (Fig. 4) (DeVries c’t al., 1970; Komatsu et ul.. 1970, DeVries et ul.. 197 1: Shier et ~1.. 1972, 1975). There are eight different sizes and the range of mol. wts is between 2600 and 34.000 (Table I). The three smaller glycopeptides differ from the larger ones in that proline replaces alanine beginning at position 7 in the glycopeptide repeating sequence until the C terminal is reached (Lin et ul., 1972. Morris et ul.. 1978). The same eight glycopeptides have also been isofated from the rock cod, G&us ogcrc, which swims beneath the ice in the near-shore waters of Labrador (Van Voorhies ct al., 1978). The same ones have also been reported to occur in the polar cod, Borcogadus saida (Osuga & Feeney. 1978). There are however slight variations in the positions occupied by proline in glycopeptide 8 in these forms compared to the Antarctic forms. The saffon cod, Eleginus gracilis, from the Bering Sea and the tomcod, Microgadus rom-

scwm

froctianwith

diolyroble other

solutes

than NaCl

Fig, 3. Frcrling points (f.p.) :md melting points (m.p.1 of the blood of II Marm%atcr temperature perch, Enrhir~toc,~r ~~K~!KSOHI. and the Antarctic fish. TWW~OI~II.S horchg~rrvirlhi. before and after extensive dialysis against distilled water. After dialysis. the freezing point of the blood plasma of the perch is - 0.01 C while that of the Antarctic fish is - I.31 C indicating that all of the freezing point depression of the former results from NaCI and orher small solutes. In the Antarctic fish half of the depression is associated with the presence of large ~n~n-~~l~~d~l~) glycoproteins or glycopepiidt%. A dialysis memhranc with a mol. wt cut-off of 3500 was used.

cod. also have glycopeptide antifreezes (Raymond it (11.. 1975). however they differ from those of the Antarctic fishes and other polar cods in several respects. Their non-colligative lowering of the freezing point of water is less and there are only three to five electrophoretic variants with most of the glycopeptide being in the form of one that has a mol. wt of approx 5000. Their compositions differ in that al1 contain some proline and a few arginine residues. Although the antifreezes in the saffron cod and rock cod are similar in that they are glycopeptides. it is unusual that they differ substantially in composition and size and are still members of the same family. Members of

three Antarctic notothenioid families all have identical glycopeptides (DeVries & Somero. 1971) indicating much less variability in the fish fauna of this ecosystem. Peptide antifreezes have been identified in and isolated from several north-temper~~te and Arctic fishes (Table 2). They vrq in size and composition and only

Table 1. Molecular weights of glycopeptide and peptide antifreezes isolated from rhe blood serum of the AntarctIc cod, ‘/i~mror~~us hort~luqrr~~idi. and the winter flounder. Pst~ttdoplr~rr~c~r~~~c~rrs ~IVW~UIIIIS, respectively Glpcopeptide or peptide No. Antarctic

giycopeptides

I

33.700 ‘8.X00 21,500 17.000

, ; 4 5

I o.soo 7900 3500 2fxh3

6 7 8 Flounder

unit of glyeopeptide antifish. The polypeptide backbone is made up of only LWO amino acids, alanine and threonine in the sequence alanyi-aianyl-threonine. Every threonine is joined to a disaccharide fi-El-galactopyrarrosyl-( 1 ----t 3f-2-acetamido-l-deoxy-a-ngalactopy~inus~ through a glycosidic linkage.

Fig. 4. Basic repeating

structural

freezes isolated from the blood of Antarcfic

Mol. wt

peptides

1 7 ;

12.000* x000* 3200

*Molecular wts estimated from relafive mobilities on SDS clectrophoretic gels and therefore probably too high. Peprides I and 1 probably have mol. wts 8000 and 5000. respectively.

8 8

18

8

Tremm~~tus hor&re~+dci,t Boreogudtts snida,f Gadrts oqac~t

* Values are number of residues per SOWg antifreeze. t DeVries, 1980. t Osuga & Feeney. 197X. BHew et al.. 1980. 11 Slaughter et al., 198I,

Tryptophan Arginine N-acetylgalatosamine Galactose

Phenylalanine Lysine Histidine

acid Threonine Serine Glutamic acid Glycinc Proline Alanine Half-cystine Methionine Isoleucine Leucine Tyrosine

Aspartic

Amino acid or sugar

7 1

1

16

3

7

Eligirttts qruci/is.t ilf icroyadus tonrrodt

I

1

2

1

1

34

I

1

1 1 1 I

1 I

1

I

1 I

1

1

1 3

I 1 3

38

3 I 3 3

-l

5 4 4 4

urnuricurnisj/

Hmifrrprrrus

31

1 2 2

I 3 I

I I

1 I 32

3 4

Mwmqh~lus srorpitrs#

3 3

~My.uocrp/~u/us mmucosust

3 6

Pltwowcte,s yIrctdrituhrrcu/lrrltst

3 6

Pscudoplelrrort~crrs ultleriru?nrst

Table 2. Comparison of sugar and amino acid content of antifreezes in coldwater fishes*

ALA-ALA-THR-ALA-ALA-T~R-ALA-ALA-THR-ALA-ALA-THR-ALA-ALA-THR-ALA-ALANiGA

NAFA

NkA

NiGA

NiGA

GAL

GAL

GAL

GAL

GAL

ASP-THR-ALA-SER-ASP-AL4-ALA-AL4-BIA-kU1--ALA-AS?A~-ALA-ALA-ALA-AL~~ALA-LEU-THR-A~-ALA-ASNAU-ALA-ALA-ALA-ALA-ALA-ALA-THR-ALA-ALA-X -

I-

4.5#-

jI

4,s

-I

ASP-THR-SEA-ASP-ALA-ALA-ALA-ALA-ALA-ALA--ASP-

Fig. 5. The primary

~truct~wr of the glycopeptides isolated from the Antarctic cod. Dissr~ficl~~ ~rr~sc~~i. The basic structural unit shown in Fig. 3 is rcpcated except m the

small glycopeptides (7 and 81 where prolmes occasional11 replace alanines. The lower panel shows the primary structure of the peptide antifreeze isolated from the blood of the winter flounder. P.\e~rdt~pl~,~rro~~~,~~f~,,\ umwicuttw. and the Alaskan plaice. P Iru~~~~it~.~. ylr~itlr.irilhrrt,lrILirlr\. In the conformation of an it helix, the aspartate and threonine residues are separated by 4.5 /S a distance that also separates the oxygens aiong the a-axes in the ice lattice.

a few have been completely

characterized. Three separate peptides have been isolated from the winter flounder. Pseudoplertl-onrctes arneric’anus. and they are composed of eight amino acids (Duman & DeVries. 1976) in which alanine accounts for 60”,, of the residues. Most of the remainder of the ones are polar residues such as aspartate, glutamate. lysine, serine and threonine. Three peptides have been isolated and partially sequenced (Fig. 5). They show a repeat pattern of the two polar residues aspartate and threonine separated by two alanines with each polar cluster separated by seven non-polar residues (usually six alanines and a leucine) (DeVries & Lin. 1977b). The Alaskan plaice. P~~,zf~(~~r~,~,f~,s yutrtlritriht,~ttfrrtlts. has peptide antifreezes which are similar to those of the winter flounder. The sequence of one shows the same polar clusters separated by long stretches of alanine. It differs in that leucine is absent, however the positions of threonine and aspartate are the same (Petzel J. & DeVries A. L.. unpublished result). Antifreeze peptides have also been isolated from the Bering Sea sculpin. M~o.~ocrplrc&f.s cerrrccosus, and there are several electrophoretic variants (Raymond et af.. 1975). It appears to be similar in composition to a peptide isolated from the short horn s&pin, M. SC’Wpius. from the waters of Newfoundland (Hew rf ill., 1980) which is not unexpected since these species are taxonomically almost indistinguishable. Like the flounder these peptides are about 60”,, alanine and rich in the polar residues aspartate. threonine, glutamate and lysine. They differ in that they contain more non-polar amino acids than the winter flounder (Raymond et ul., 1975). Recently an antifreeze peptide has been isolated from the sea raven, ~e~~zjt~j~fe~~~ nmericumfs. which differs substantially from the other pep-

tide antifreezes in that it has reduced amounts of alanine and the presence of relatively large amounts of glycine and some of the aromatic amino acids (Slaughter et ctl.. 1981). The presence of the aromatic amino acids and glycine is unusual as none of the other peptide antifreezes contain them and an explanation for their functional role is awaiting. The only Antarctic fish that possesses a peptide antifreeze is the eel pout, ~~1~~(~p~7;~~ ~~~~~bo~~7i. and it has 12 amino acids much of which is alanine (DeVries. 1980). Studies of these few fish antifreezes have revealed that they arc either glycopeptides or peptides and there appears to be considerable variation in some cases and similarities in other cases in regards to their composition and structure. In some cases fishes belonging to unrelated families inhabiting opposite hemispheres have evolved the same antifreeze glycopeptides (DeVries et uI.. 1971: Van Voorhies c’r (11.. 1978) while others which are sympatric species of the same family and therefore more closely related have evolved glycopeptide antifreezes which show considerably more variation in the amino acid composition. Only limited information about the secondary structure of the antifreezes exists. It is known from studies involving dialysis. viscosity and circular dichroism measLlrements that both the glycopeptide and peptide antifreezes are expanded molecules (DeVries et (I/.. 1970; Raymond et (I[.. 1977: Franks & Morris. 1978). X-ray diffraction, detailed circular dichroism studies and natural abundance C-13 nuclear magnetic resonance studies have not given definitive information on the secondary structure of the glycopeptides (Ahmed rf al., 1975: Raymond rt uI.. 1977; Franks & Morris, 1978; Berman er ul.. 1980: Bush et ul.. 1981). Circular dichroism studies and viscosity measurements (Raymond tif tri.. 1977; Ananthanarayanan & Hew. 1977) mdlcate that most ot the peptide antifreezes are in the form of rigid rods in a completely helical conformation. The significance of this conformation is that the polar residues which are separated by two alanines are also scparatcd by a distance of 4.5 8, (DeVries & Lin. 1977b). The oxygens in the ice lattice are also separated by 4.5 A (Fletcher. 1970). This repeat spacing of the polar residues and oxygens results in a lattice match which appears to hc of’ paramount importance for recognition and binding of the peptide to the ice lattice.

NOh-(‘OLLIGfrTIVE LOWERING OF THE FREEZING POIYT

The unusual freezing behavior of the blood of polar fishes has lead several investigators to study the freczing--melting behavior of the blood and solutions of peptide and giycopeptide antifreeze (DeVries. 1971: Scholander & Maggert. 1971: Raymond, 1976: Raymond & DeVries, 1972. 1977: Mulvihill et (II.. 1980: Tomimatsu er (I/., 1976). These studies have included freezing point and m.p. estimates determined by a variety of techniques. The technique of choice. which encompasses thermodynamic considerations, involves observations of melting and the growth of a microscopic seed ice crystal as the temperature is slowly raised or lowered (0.01 C/min) (DeVries. 1971). The

Antifreeze agents in coldwater crucial factor in this technique is that the variable element of supercooling is eliminated as it has a tremendous effect upon the observed freezing point (Raymond & DeVries, 1972; Osuga rt al., 1978). To most people, the “freezing point” is the temperature at which a solid is in equilibrium with its liquid and therefore freezing point and m.p. are assumed to be the same. When samples of plasma containing antifreeze or solutions of the peptide and glycopeptide antifreezes are cooled below their m.p. in the presence of a seed crystal, no crystal growth is observed until the temperature is substantially lowered. In a 27, solution of antifreeze, growth does not occur until the temperature is lowered to - l.l’C and then it is very rapid and in the form of long spicules whose direction of growth is parallel to the c-axis. the nonpreferred axis of growth (Fig. 6). The spicular ice that is formed. as well as the seed crystal. will not melt until the temperature is raised to - 0.02’.C, a temperature determined by the solution’s colligative properties (Raymond. 1976). The size of the seed crystals at temperatures intermediate to melting and freezing temperatures appears to be stable (Raymond & DeVries, 1977). For the most part all of the glycopeptide and peptide antifreezes exhibit the same depression of the freezing point of water on a weight basis, except for some of the smaller glycopeptides isolated from the Antarctic Nototheniids (Lin et al., 1972) (Fig. 6). The freezing point of a 4”/, antifreeze solution is - 1.2”C while the m.p. of the ice in it is approx - 0.02”C. However if their abilities to lower the freezing point are plotted on a molar basis then it appears that the freezing point depression of water is correlated with size. with the larger ones showing a greater effect than the smaller ones (Schrag et ul., 1982). If freezing point estimates are obtained by techniques where the element of supercooling enters in then much different estimates of the freezing point are obtained (DeVries. 1974). If the freezing point osmometer in which supercooling by 4~C leads to rapid FREEZING-MELTING BEHAVIOR GLYCOPEPTIDE 8 PEPTIDE ANTIFREEZES

freezing of the samples is used, then not only do the large mol. wt peptides show reduced freezing point depressions but the small ones show only that depression of the freezing point expected on the basis of colligative relationships (DeVries, 1974). When small amounts of the large glycopeptides (109,) are mixed with the small glycopeptides and the freezing point of such solutions estimated on the freezing point osmometer. then a large potentiation of the antifreeze activity of the small glycopeptides is observed (Osuga et uI., 1978). However when the freezing points are determined in the presence of a seed crystal with the system closely approaching thermal equilibrium (temperature changes of only O.Ol’/min) then no potentiation of the freezing point depression is observed (Schrag J. D. & DeVries A. L., unpublished results). In such studies. prevention of supercooling prior to freezing is of the utmost importance because the freezing points obtained in this manner give results that can be very misleading. It is obvious that fishes living even in the most extreme environments such as the Antarctic Ocean experience not only small temperature changes. but ones that take place very slowly (Littlepage. 1965). The reason for this is because of the high heat capacity of water and the large heat of fusion associated with freezing. Thus freezing points determined with slow changes in temperature more closely approximate what a fish would experience in nature. In this context one question that needs to be addressed is whether freezing points of the blood are representative of the lack of freezing or freezing avoidance in the fishes. Thus far ice has never been observed in the polar fishes in nature. Neither have fishes been observed to exist in a partially frozen state; if partially frozen they die. In this context the relationship of freezing point to freezing avoidance needs to be reconciled. Using the m.p.-freezing point technique it can be shown that most of the glycopeptide and peptide antifreezes exhibit nearly the same non-colligative lowering of the freezing point of water except as mentioned above for some of the smaller glycopeptides present in the blood of the Antarctic Nototheniids. The mechanism by which these antifreezes produce this unusual depression of the freezing point of water is not fully understood at this time and is currently being investigated by several laboratories.

q/'nor~-colligatic~r ,f~re_ing

Mrchmism

(mg/mll

poinf

drpressiorl

Since the antifreezes have expanded structures (DeVries ct al., 1970: Raymond & DeVries, 1977: Raymond rt ul.. 1977; Ahmed rt nl.. 1975; Franks & Morris, 1978) and since they have side chains that are rich in hydroxyls or rich in polar groups. it seems that they might be good candidates for structuring water about themselves. Recent studies employing nuclear magnetic resonance techniques (Haschemeyer rt ul.. 1977) indicate that the amount of water that is bound is small. Isopiestic determinations of water binding under equilibrium conditions indicates that they bind only slightly more water than other proteins of a similar size when in solution (Duman ~‘r al., 1980). It would appear that the small amounts of water that are “bound” are much too small to explain the antifreeze effect. Lack

Fig. 6. Freezing point and m.p. of aqueous solutions of glycopeptide and peptide antifreezes. On a weight basis the freezing points of glycopeptides l-5 are approximately twice those of glycopeptides 7 and 8. The freezing points of solutions of the peptide antifreezes isolated from the flounder and sculpin are approx the same as those of the large glycopeptides. The m.p. are the same for all of the antifreezes. Freezing point and m.p. were determined as described in the text.

633

fishes

tf

r~%fenw

jtir

~~~frr

structwitug.

634

AIUHUR L. DFVKES

Non-colligatiw iowering qj’ the ,fkerziny point through u~sorption-inhibition Adsorption. In the field of crystal growth and inhibition of crystal growth there is considerable information that indicates that adsorbed impurities can inhibit the crystallization or the growth of small crystals (for review see Raymond, 1976). Such inhibitors are usually characterized by a specificity for a particufar kind of crystal and the large molecules with repeating units are more effective than small ones. It is currently thought that adsorption of an impurity inhibits crystal growth by interferring with the propagation of steps across the face of the crystal. fn many cases, adsorption of impurities also causes a change in the type of crystal growth. or habit, that is observed when the supersaturation point is exceeded (Buckley, 1952; Butchart & Whetstone. 1949). The inhibition of ice crystal growth below the equilibrium freezing point of water by the glycopeptide and peptide antifreezes appears to be another example of the adsorption-inhibition phenomenon. Studies of freezing behavior of solutions of the glycopeptide and peptide antifreezes indicate that they adsorb to ice (Duman & DeVries. 1972: Tomimatsu c’t (II., 1976: Raymond & DeVries. 1977). The affinity for ice varies with the mol. wt of the antifreeze with the smaller molecules showing less binding than the larger ones (Raymond & DeVries. 1977). The affinity for ice is lost if the antifreezes are chemically modified (Raymond & DeVries. 1977) as well as their antifreeze activity (Shier pt al., 1972; Duman & DeVries. 1971). In the case of the glycopeptides. alteration of the hydroxyls of the carbohydrate moiety leads to loss of activity (Lin rt al.. 1976; Duman & DeVries. 1972) as well as limited cleavage of the polypeptide backbone (Komatsu et al.. 1970). Reduction in size of the glycopeptides by sequential degradation results in decreased binding and antifreeze activity (Schrag it rii., 1982) which is in line with observations that large repeating polymers are better inhibitors of crystallization than small molecules (Raymond. 1976). With the peptides. modifications of the carboxyl groups of aspartic and glutamic acid residues result in loss of activity (Duman & DeVries, 1976) and presumably binding. Specific modification of the sculpin peptide antifreeze by attachment of fluoroscein to its four lysines results in complete loss of activity (Scbrag J. D. & DeVries A. L.. unpublished results). Loss of antifreeze activity, resulting from moditication of polar side chains. appears to he correlated with a loss in the affinity for ice. Since all of the polar side chains are potential hydrogen bonders. it appears that the antifreezes probably recognize and bind to ice through hydrogen bonding. In order for the hydrogen bonds to be as strong as possible. the potential hydrogen bonding residues should be located in the molecule in positions such that they would be aligned with the oxygens in the ice lattice. If ail the polar residues are positioned so that they can partioipate in linear hydrogen bond formation with the oxygens (or hydrogens) of the ice lattice. then the binding to ice would be maximized. A distortion of the hydrogen bond angle would probably lead to reduced binding and reduced activity. Examination of space-filling models of the glyco-

peptides reveals that many of the hydroxyls of the disaccharide side chain are spaced 4.5 A apart, a distance that separates the oxygens in the ice lattice parallel to the a-axes. Although the secondary structure of the glycopeptides remains to be elucidated (Berman ~lt [I/., 1980; Bush ~‘rtrl.. 19XI ) it is instructive to speculate how various conformations might recognize and bind to ice. It is possible that the carbohydrate moieties function to keep the polypeptide of the antifreeze in :I completely extended conformation. Such a conformation is attractive because alternate carbonyl groups project from the same side of the polypeptide and in the extended ~onforn~~~tion they are separated by 7.3 A (Pauhng rat tri,. 1951). This distance also separates alternate oxygens along the c-axis in the ice lattice (Fletcher, 1970). This 7.36 A spacing of the oxygcns in the Iatticc is ;I repeat spacing. Some evidence for the existence of :I completely extended conformation exists (Franks & Morris. 197X) but is not overwhelming. Sequence studies of the flounder antifreeze pcptidc indicate the presence of clusters of polar amino acids separated by long sequences of non-polar &nine residues. The polar clusters usually contain threonine and aspartate separated by two alanines (DeVries & Lin. 1977b). In the Alaskan plaice antifreeze peptidc II similar polar. non-polar arrangement is also present. Physica- chemical studies indicate all these peptides are in the form of an x-helix (Raymond c’t ril.. 1977; .i2nanth;lnaryanan & Hew. 1977). In such a ~Onforrn~Iti~~n, the polar side chains are located on one side of the helix while the nonpolar sides chains ;Ire on the other. Measurements of the distance between aspartate and threonine in such a conformation shows the! arc separated by 4.5 A. a repeat distance that also separates adjacent oxygens in the ice lattice parallel to the :I-;IXL”S.This lattice match between the polar residues and the oxygens in the ice lattice suggests that the peptides orient thomselves on the ice lattice and bind to it through hydrogen bonding. Fipure 7 sho\vs how the peptides prohably hydrogen-bond to ice. In such ;I model every third row of oxygens in the ice lutticc is not involved in bonding. This may be important bc:uause an uninterrupted 4.5 il rcpcat spacings of the polar residues could possibly make the antifreezes behave as a nuclCntor. The complete sequence thus far has been determined for one antifreeze peptide in the flounder and Alaskan plaice. In the latter the repcat spacing of the polar threonines and aspartates is conserved. and their positions are the same. Preliminary sequences of the sculpin peptides also indicate the presence of threonine or serine separated by 3.5 A from either aspartate or glutamate (Schrag J. D. & DeVries A. L. unpublished results). Recentlv it has been suggested that the polar clusters participate in [I’ turns between the helical segments of alanine (Loucheux-LeFebvre. 1978). In such turns a 4.5 A spacing between aspartate and threonine could still be conserved. however recent fiuorescence polarization studios indicate that the peptides are rigid rods and therefore probably lack /I turns. Further studies are in order to verify the underlying structural requirements necessary for binding to ice. I~l~ihitim. Anomalously low freezing points of water’ in gels and tissues have been explained on the

Antifreeze

agents

in coldwater

635

fishes

MODEL OF PEPTIDE ANTiFREEZEHYDROGENBONDED TO ICE

I

i HYDROGEN BOND--'

ICE LATTICE--,

0

o'

0

1 lo;I

g

g I

0

0

0

0 A-AXIS-

0

o,5Ay

r*

0

0

I

Fig. 7. Model of winter flounder peptide antifreeze hydrogen bonded to the face of an ice crystal parallel to one of the a-axes. The circles represent oxygens in the ice lattice and the darkened ones represent those that participate in hydrogen bond formation with the hydroxyl of the threonine residue and the carboxyl group of aspartate. These two residues are separated by 4.5 A, a distance that also separates the oxygens in the ice lattice parallel to the a-axes.

basis of increases in surface free energy resulting from a high ratio of surface area to volume. In gels. water is assumed to be able to freezeonly in the form of microcrystals which have a large surface area relative to volume (Block et al.. 1963; Kuhn. 1956). The increased surface relative to volume raises the surface free energy. In order for freezing to occur then, energy must be removed from the system. This is done by lowering the temperature. The appearance of crystals then at a lower temperature would be the same as stating that the freezing point of water was lowered (Kuhn, 1956). Adsorption of the antifreezes to ice crystals could conceivably lead to an increase in surface area with only a small increase in volume and result in a lowered freezing point. The evidence for this hypothesis and the mathematical analysis are given in detail elsewhere (Raymond, 1976: Raymond & DeVries. 1977). Here we give only a qualitative overview, in which adsorption of the antifreeze leads to an increase in surface free energy. Assuming that ice crystal growth occurs via water molecules joining the crystal on the basal planes at steps, then adsorbed antifreeze molecules would force growth to occur at the step only in the areas in between them. As a consequence the step would be divided into many fronts separated by adsorbed antifreeze as shown in Fig. 8. These fronts will have a large surface area compared to their volume because they have highly curved fronts. The small distances between the adsorbed antifreezes leads to the growth of highly curved fronts. Growth between the molecules will stop when the ratio of surface area to volume exceeds a critical point. This is related to the radius of curvature of the front and when it is equal to one half of the spacing between two adsorbed adjacent antifreeze molecules. growth will stop. If the spacing between the antifreeze mol-

ecules is reduced then the undercooling required to allow the step to propagate through the spacing must be increased. Another way of stating this is that the freezing point of water is lowered. The spacing between the antifreeze molecules appears to be a function of their concentration. size and shape. If certain assumptions are made about the density and randomness of the antifreeze molecules on the crystal face, it can be stated that the undercooling (or freezing point depression) is proportional to the square root of the concentration. Using this relationship it can be shown that there is good agreement between freezing point depression curves obtained experimentally and those derived from the above relationship (Raymond & DeVries. 1977). DISTRIBL~TIOIV OF ANTIFREEZE BODY FLL’IDS

IN

The antifreezes present in fishes exist in a number of forms which vary in size and also slightly in composition (DeVries. 1980). In addition antifreezes within a given species show different activities with regards to their freezing point depressing activities (DeVries. 1974). It is not clear why the many forms exist and whether they all play a role in protection of the body fluids from freezing. In the Antarctic fishes the eight glycopeptides are present in the blood, pericardial fluid. coelomic fluid, cerebral spinal Ruid. however none are present ‘in the urine. The large mol. wt forms are not present in the intracellular fluid of the fishes. however it appears that a small amount of the low mol. wt forms are present within the cytosol. It would appear that they might be important for prevention of freezing of cells which make intimate contact with the small ice crystal. The gills of fishes are often bathed with water that

636

ARTHUR L. DEVRIES

DIRECTION OF C AXiS

Fig. 8. A model ill~istr~tin~ the adsorption inhibition as a mechanism of non-colii&~ti~e lowering of the freezine ooint of water. The antifreeze molecules arc adsorbed on the face of the crystal at the step. The bound%tifreere molecules force water molecules. in this case represented by cubes. to join the ice lattice between the adsorbed molecules resulting in growth in the form of areas having ;1 high radius of curvature. In order for ice to grow in an area of high curvxture. the temperature must he lowered. Stated m another way It can he said that the freezing point is lowered.

is laden with minute ice crystals yet they appear not to freeze. Intracell~ilar glycopeptide could conceivably result in protection. Recent studies have shown that the intestinal fluids of polar fishes contain antifreeze peptides and glycopeptides (O’Grady, t’r ~1.. 1982b) and that they are present in quantities that lower the freezing point of this fluid below that of seawater. In the Antarctic fishes the mtestinal fluid is fortified with only the low mol. wt forms. glycopeptides 6. 7 and 8. Their presence in the intestinal fluid is necessary to prevent freezing for the following reason. Marine fishes drink seawater to maintain water balance which is accomplished by absorption of NaCI and water as it passes along the intestine. Absorption of about half of the salt and water renders the intestinal fluid isosmotic to the blood. As a consequence the freezing point I C‘ above the rises to - I -‘C which is approx ambient temperature of the fish. As the :tnal sphincter is rather open to envir~~nrneilt because of :I “loose” sphincter ice crystals could conceivably propagate through the opening and lead to freezing of the super-

cooled intestinal fluid if it were not for the presence of the glycopeptide antifreeze. The concentration of glycopeptides 6. 7 and 8 appears to be high enough in the posterior portion of the intestine so that the freczing point is lowered to -- 1.2 C. a temperature well below the temperature of the seawater. The bile in the gall bladder is also fortified with these glycopeptides. It appears that the glycopeptides find their way into the intestinal fluid when the bile is expelled into the anterior end of the duodenum via the cystic duct. As food and water are digested absorbed. the glycopeptides appear to remain intact and are concentrated to levels where they lower the freezing point of the intestinal fluid to - 2.3 C. So far there is no evidence that any of the glycopeptides are digested or reabsorbed as they pass down the digestive track and thus they are probably excreted. Their loss might appear wasteful from an energy point of view. however prevention of freezing of the intestinal fluid is the difference between living and freezing and in the broad picture the loss of some glycopeptide antifreeze is an energy loss necessary for survival.

Antifreeze

agents

Although cold water fishes commit the loss of some antifreeze to ensure that their intestinal fluid does not freeze there are other mechanisms which ensure that these molecules are conserved within the circulation. The urine of such fishes lack antifreeze and mechanisms exist for their conservation which will be discussed shortly. The absence of antifreeze in the urine of polar fishes results in the appearance of supercooled urine in the bladder (Dobbs et al., 1974). Threasons why such urine, especially in the case of the Antarctic fishes, can exist in a supercooled state for the entire life of the fish is not entirely clear. It could be argued that the one degree of supercooling is metastable for the life span of the fish (100 years in some cases). If this is the case, then the body wall which is fortified with antifreeze prevents entry across the body wall. It is not clear why ice does not propagate up the uretha during micturition or thereafter. Perhaps the rate of ice propagation up the urine stream occurs too slowly and when the sphincter is tightly closed. the mucous lining the sphincter wall may inhibit propagation and prevent entry. These questions novel approaches to provide plausible require answers. RENAL

CONSERVATION AND PEPTIDE

OF GLYCOPEPTIDES ANTIFREEZES

The kidneys of many marine teleosts are composed of glomerular nephrons that freely filter inulin and polyethylene glycol which are glomerular markers used for determining glomerular filtration rates (Hickman & Trump, 1969; McKenzie et al., 1977). The mol. wts of these markers are approx 5000. which is similar to the mol. wts of many of the glycopeptide and peptide antifreezes. Most of these antifreezes are small enough so they should be filtered into the urine at the same rate as PEG. Urine m.p.-freezing point and radioimmunoassays specific for the antifreezes indicate they are not present in the bladder urine (Dobbs & DeVries 1975a: DeVries & Lin. 1977a; Petzel & DeVries. 1979. 1981). The reason that the glycopeptide antifreezes are not present in the urine of the Antarctic notothenioid fishes is that the kidneys are made up entirely of aglomerular nephrons (Dobbs rt ctl.. 1974: Dobbs & DeVries. 1975a,b). With these fishes. urine formation is the result of secretory processes (Dobbs & DeVries. 1975a.b). A few of the true cods in the northern hemisphere such as Gadus r,yclc have kidneys in which some glomerular nephrons are present but they also lack antifreeze in their urine. Their absence appears to result from the fact that their glomeruli are non-functional. Histological examination shows reduced numbers of capillaries in the glomerular tuft and a thickened basement membrane which results in an increased filtration barrier (Eastman J. T. & DeVries A. L.. unpublished results). In many of the northern fishes that have peptide antifreezes. functional glomeruli are present although in fewer numbers than in higher vertebrate kidneys. Some of these fishes freely filter inulin and polyethylene glycol (Mackenzie of [I/.. 1977; Petzel & DeVries, 1979) and the rate of filtration is of such a magnitude that if the antifreeze peptides were filtered they would be eliminated from the circulation in just a few days. Although extensive studies have not been done on

637

in coldwater fishes

such fishes. it appears that the antifreeze peptides are conserved in the circulation of the winter flounder because they are not filtered (Petzel & DeVries, 1980. 1981). The reason they are not filtered is because they are repelled from the basement membrane of the gIomerular capillary wall by a charge repulsion mechanism. The isoelectric points of the antifreeze peptides in the winter flounder blood are between 4 and 5 and the basement membrane appears to have a negative charge as judged by its ability to bind cationized ferritin (Petzel D. & DeVries A. L. unpublished results; Boyd & DeVries, 1982). The negatively charged components of basement membrane probably create a negative electric field which repells the negatively charged antifreezes from the pores in the glomerular capillary wall. Cationization of the antifreeze peptides leads to a large increase in their clearance suggesting that the native anionic form of the antifreeze is conserved in the circulation by such a charge repulsion mechanism (Petzel & DeVries. 1981). In many of the other fishes which possess peptide antifreezes. the kidneys are made up of some glomerular nephrons. Conservation of the antifreeze in the circulation in these fishes is due to the fact that their glomeruli appear to be non-functional. The Antarctic eelpout. Rigophiliu drarhorni. which lives in McMurdo Sound has an acidic peptide antifreeze which is needed for survival throughout the year because the Sound is always at its freezing point. The peptide is conserved in the circulation because the number of glomeruli have been reduced and those that are present do not filter inulin as they have a very much thickened filtration barrier (Eastman et ul.. 1979). Maintenance of high levels of antifreeze throughout the year is necessary in this fish for survival and elimination of filtration would appear to be the simplest means for conservation of the antifreeze. Other fishes such as the short horn sculpin. M. scorpius, also appear to have reduced the number of glomeruli in their kidneys compared to closely related species such as M. octodecemspinosus which lack antifreeze. The few nephrons that are present appear to be non-functional. The black cod, Notohmia anyustutu. lacks an antifreeze as it spends its entire life in the temperate waters of southern New Zealand. This nototheniid lacks antifreeze but is pauciglomerular. It therefore appears that a correlation exists between aglomerularism and the presence of glycopeptide and peptide antifreezes in many of the polar fishes. VARIATION

OF ANTIFREEZE

LEVELS

WITH SEASON

Compared to the high Arctic and Antarctic, northtemperate environments experience large seasonal temperature fluctuation. As a consequence species like the winter flounder require antifreeze and synthesize it only during the cold months (Duman & DeVries. 1974a,b; Fletcher. 1977; Petzel rt (II., 1980; Fletcher. 1981). It is synthesized by the liver during the late autumn and it appears that the control of antifreeze biosynthesis occurs at the level of transcription and possibly translation (Lin, 1979; Davies & Hew, 1980; Lin & Long, 1980). Antifreeze messenger RNA is present in September about a month before any peptide can be detected in the blood. Messenger disap-

638

ARTHUR L. DFVRII:S

pears in March, a month or two before antifreeze disappears from the blood (Lin. 1979; Davies & Hew, 1980). Disappearance f.om the blood appears to depend upon temperature. The environmental-factors that lead to seasonal changes in levels of antifreeze would appear to be temperature and photoperiod. Warm acclimation studies during the early winter leads to a reduction in blood levels of antifreeze (Duman & DeVries. 1974b). A possible interpretation of these observations is that at the high temperatures antifreeze synthesis may be continuing to occur but the degradation may be accelerated more than synthesis resulting in reduced levels of antifreeze in the blood (Fletcher. 198 1). Preliminary studies involving warm acclimation experiments on winter specimens with short and long photoperiods suggested that short photoperiods caused retention of the antifreeze (Duman & DeVries. 1974b). Recent extensive studies however demonstrate that photoperiod has no effect on the disappearance of antifreeze in the spring (Fletcher. 19X1). Long photoperiods in the autumn delay but do not prevent the appearance of the antifreeze (Fletcher, 1981). From recent studies it would appear that the annual antifreeze cycle may be endogenously controlled (Fletcher & Smith. 1980; Petzel et ul.. 19X0; Fletcher. 1981). Evidence for endogenous control can be found in the fact that temperature and photoperiod have so little influence on the cycle (Fletcher. 1981). Support for this hypothesis also comes from the fact that flounder from Nova Scotia maintained their antifreeze cycle characteristic of Nova Scotia when transferred to Newfoundland (Fletcher & Smith. 1980). The appearance of antifreeze production in the autumn appears to be correlated with gonadal devclopment. Removal of the pituitary gland results in maintenance of antifreeze levels in the summer that are comparable to winter levels (Fletcher et ctl.. 1978) which could be interpreted as evidence for the antifreeze cycle being tied to the reproductive cycle. It is quite possible that the cycle is closely linked to gonadal development and spawning. If this is the case the turning on and off of the antifreeze cycle may be controlled by levels of steroid hormones elaborated by the testes and ovary. The control of the annual antifreeze cycle is an area of research which deserves further attention. In contrast to the variable north-temperate environment. the waters of McMurdo Sound arc at their freezing point throughout the year. Antarctic T~c~rriclr(~~~~~r,~ fishes therefore always require a protective antifreeze. Although these fish are extremely stcnothermal (Somero & DcVries. 1967) they can bc warm-acclimated for long periods of time at + 4 C. After 60 days at + 4 C. the blood level of glycopeptide antifreeze appears to remain unchanged. In healthy feeding fishes the half-life of the antifreeze may be ItJOdays (DeVries. unpublished results) and therefore 60 days of acclimation may be insufhcicnt to observe much of a decrease in the antifreeze level in these species. However. during warm acclimation antifreeze messenger RNA remains constant and at + 4 C liver hepatocytes continue to secrete antifreeze glycopeptide (O’Grady et cd.. 1983a) suggesting temperature has little influence on control of synthesis in this system.

Other Antarctic fishes living in warmer environments have lower levels of antifreeze (DeVries & Lin. 1977a) suggesting that the antifreeze level has been adjusted over evolutionary time in response to temperature. It appears that temperature has an effect on levels of antifreeze present in species inhabiting differing thermal environments but does not have an effect upon the level in an individual Antarctic species. These findings suggest that some of the Antarctic fishes have lost their capacity to acclimate to thermal change. The reason for this lack of response is unclear but perhaps because of the long-term stability of the Antarctic marine environment the need for Antarctic fishes to maintain genetic variability for acclimation to differing thermal environments was lost (DeVries. 197X). .-1c~lirlol~lcll~c~t~l~t1t.s Much of the research described above was supported by the National Science Foundation grants PCM 77 25166 and DPP 7X-23462 to ALD. REFERENCES L. s.. ~-EENFY R. F.. OSUGA D. T. & YtH Y. (1975) Antifreeze glycoproteins from antarctic tish. _I. hiol. Clwrx 250. 3344 3347. AUAN.rHANAR1.AXAN V. S. & Hbw C. L. ((977) Structural studies on the freezing point-depressing protein of the winter flounder Pst~rtdoplt~~tro~~~~~~t~~.\ III~~~~~~~uI~IS. Bi~dWftl. hio/~h,w. Ru.\. C‘rw~mun. 74. 685~ 689. ANIIRIASHEL. A. P. (1970) Cryloplagic fishes in the Arctic and antarctic and their significance in polar ecosystems. In .Amrttrr’c’/i~~ hhx/~~. Vol. 1 (Edited by HOLIIGAT~ M. W.I pp. 297 30-t. Academic Press. London. Bt RMAN E.. ALLrRHANn A. & DI~VRI~S A. L. (1980) Natural ahundancc carbon 13 nuclear magnetic resonance spectroscopy of antifreeze glycoproteins. .I. hiol. Clwrn. 255. 4407~_44 10. BLAC h: V. S. (195I) Some aspects of the physiology of fish. II. Osmotic regulation in teleost fishes. L’nir. 7;wortro Stutl. hiol. Ser. .CV71, 53-89. BI och: R.. WALIIK D. J. & K~IHN W. (1963) Structurally caused freezing point depression of biological tissues. J. <,(‘)I.f’h),\iol. 46. 605 6 15. Bo\ 11 R. B. Rr Dr.V~urs A. L. (1982) Localization of anionic binding sites on the glomerular basement membrane of a cold water teleost. Proc. 40th .AwI. MC/ Elrctrm Micrc !CCl/J!’sot,. .4ni.. pp. IO I I. Bt cx~t Y H. F. (lY51) Crj.stu/ C;r~nc.rh. p. 339. Wtley. New York. AHhlro

BUSH C. A.. FI:I.NI y R. E.. OSLIGA D. T.. RALA1’A.U S. & YI FI Y. (19X1) Antifreeze glycoprotcin. J. P~~ptrd~~ froth

RCA. 17, 135 129. BUIC.HART A. & WHETSONE J. (1949) The effect of dyes on

crystal habits of some oxy-salts Discuss Furuduy Sot. 5. 254 261. DA\ II s I’. I.. & Ht.u C. L. (1980) Isolatron nnd characteriziuion of the antifreeze protein messenger RNA from the winter flounder. J. hiol. C~CWI.255, X729 X734. DAYTON P. K.. ROHHILLIAKUG. A. & DFVKI~S A. L. (1969) Anchor ice formation in McMurdo Sound. Anatarctica. and its biological effects. Scimr~c, 163, 273 274. D~VRI~S A. L. (196X) Freezing resistance in some Antarctic fishes. PhD Thesis. Stanford University. Stanford. California. DI-VRI~+ A. L. (1971) Glycoproteins as biological antifreeze agents in Antarctic fishes. S~~icncc 172. 1152-l 155. DI.VRIf:S A. L. (1974) Survival at freezing temperatures. in Biochcwic~trl /cx/I’. V’nl.

trrd

Biopk!~vlctr/ Peywtirr5 it! Murinv BioSARNNT J. S. & MALI-INS D. W.)

I (EdIted hy

pp. 7XY .330. Academtc

Press. London.

Antifreeze

agents

in coldwater

fishes

639

FLETCHER G. L.. CAMPBELL C. M. & HEW C. L. (1978) The DEVRIE~ A. L. (1978) The physiology and biochemistry of effects of hypophysectomy on seasonal changes in low temperature adaptations in polar marine ectoplasma freezing-point depression, protein “antifreeze”. therms. In Polur Rr.~rurch IO the Prom ud the Fmrr and Na’ and Cl- concentrations of winter flounder (Edited by MCWHINNIF M. A.) pp. 175-202. Westview f P.seud(~p~~z~~~?~~~~~s utnrricanus). Cm. J. Zod. 56, Press. 109.. i 13. DEVKIES A. L. (1980) B~ologic~~l antifreezes and survival in freezing environments. In Afrirt&s trtid ~/1rir(~}i1~~~1ir~4~ FLETCHER G. L. & SMITH .I. C, (19X0) Evidence for permanent population differences in the annual cycle of plasma Fitrwss (Edited by GILLES R.) pp. 583-607. Pergamon “antifreeze” levels of winter Rounder. Can. J. Zoo[. 58. Press, Oxford. 507 512. D~VRII:S A. L. & EASTMAN J. T. (1982) Physiology and FRANKS F. & MORRIS E. R. (1978) Blood glycoprotein from ecology of notothenioid fishes of the Ross Sea. J. RI Sot. Antarctic fish. Biochim. hiop!rJ’s. Actor 540, 34&3X. N.Z. 11, 329-340. HAS~H~M~YER A. E. V., G~s~H~~A~~R W. & DI.VKIES DEVHIES A. L.. KOMATSU S. K. & FI:~uF\I. R. E. (1970) A. L. (1977) Water binding bv antifreeze nlvcooroteins Chemical and physical properties of freezing pointfrom .i\ntarctic tish. A’trmrr. L&J. 269. 87-ii L depressing glycoproteins from Antarctic fishes. J. hiof. HEW C. L., FLETCHER G. L. & ANANTHANAKAYANAN V. S. Chot~ 245, 2901 -2913. (1980) Antifreeze proteins from the shorthorn sculpin. DEVRI~.S A. L. & LIN Y. (1977a) The role of glycoprotein Mpo.uo~c,pkuhts scorpitcs: Isolation and characterization, antifreezes in the survival of antarctic fishes. In .4&pGun. J. BirX&m. 58, 377-383. turiom withitr .4mcwric Ecos~sret~s (Edited by LLANO HICKMAN. JK.. C. P. & TRUMP B. F. (19691 The kidney. In G. A.) pp. 439-458. Gulf. Houston. Texas. F&/I F~I~.s~~J/~~~~. Vol. I (Edited by HOAR W. S. & KANDEVRIES A. L. & LIN Y. (1977b) Structure of a peptide DALL D. J.) 91-239. Academic Press. New York. antifreeze and mechanism of adsorption to ice. Biochim. KOMAIX, S. K.. D~VKIES A. L. & FEFNI’V R. E. (1970) hiophys. Acts 495, 388-392. Studies of the structure of freezing point-depressing DEVRIES A. L. & SOMFRO G. N. (1971) The physiology and glycoproteins from an antaractic fish. J. bid. Chtw~. 245, biochemistry of low temperature adaptation in antarctic 290 i-790x. marine organisms. In Antrrrcric lcr and %ft*r i%f~lssrs K~IHN W. (I9561 Uber die durch anomale krjstallgestalt (Edited by DEACON G.) pp. 101-113. Committee on sowie durch limitierung der Kristallgrosse bedingte Antarctic Research. gefrierpunktserniedrigung. Heir. &,,I. .4cta 39, D~VRIES A. L.. VANDENHEFUE J. & FEENEY R. E. (1971) 1071-1086. Primary structure of freezing point-depressing glycoLEIM A. H. & SCOTT W. B. (1966) Fish of the proteins. J. hid. C/wm. 246, 305 308. .4thric Co~rsr o/‘Crrr~udu. p, 357. Ottawa, Fish. Res. Bed. D~VRIFS A. L. & WOHLSTHLAG D. E. (1969) Freezing resistCan. ance in some antarctic fishes. Science 163. 1074-1075. LIU Y. (1979) Environmental regulation of gene expression. Dorms G. H. & D~VRII:S A. L. (197%) Renal function in J. Rio!. Chc~tn. 254, 14X-1426. antarctic teleost fishes: Serum and urine composition. LIN Y.. DU.MANJ. G. & DEVRIES A. L. (1972) Studies on the !Llcrr. Bicrl. 29, 59 70. structure and activity of low molecular weight glycoDones G. H. & D~VRII:S A. L. (l975bl Aglomerular nephproteins from an antarctic fish. Bioc~htvn. bioplly,\. Rrs. ron of Antarctic teleosts: A light electron microscopic Cmrtt~m. 46, 87-92. study. Ti.s.suc rrnd Crll 7. 159- 170. LIU Y. & LONC D. J. ll9HOl Prufication and characterizDorms G. H.. LIN Y. & DEVKI~,S A. L. (1974) Aglomrrularation of winter flounder antifreeze peptide messenger ism in Antarctic fish. SL.~PIZ~C~ 185, 793 794. ribonucleic acid. B~~~~~I~,/?I~s~~~ 19. I1 1l-l 116. D~:MAN J. G. & D~VRIES A. L. (1972) Freezing behavior of LIN Y.. RAI.MONU J. A.. DUMAN J. G. & D~VKIES A. L. aqueous solutions of glycoproteins from the blood of (1976) Compartmentalization of NaCl in frozen solantarctic fish. Crwbioloqy 9, 469-472. utions of antifreeze glycoproteins. Cryrthioloy~ 13. D~IWAN J. G. & DFVKI~S A. L. (1974a) Freezing resistance in winter flounder. Psclrdoplc~il,o~~~,~,~~s ~1~tIt~~ic~ii2u.s. 334 340. LI.TTLFI”AC;EJ. L. 11965) Oceanographic investigations in Nattlr-c. Lotid. 247, 237 -238. Mc~urdo Sound, Antarctica. In .4ntorcric, R~~.\~,ur~,~7 DLIMAN J. G. & D~VKIES A. L. Il974b) The eifects of temScrics. lid. 5. Bioiogy f$ .4nfutctic &OS If pp. 1 37. perature and photperiod on the production of antifreeze (Edited by LLANO G. A.) American Geophystcal Union. in cold water fishes. J. c’zp. Zooi. 190, X9-97. Washington. DC. DLIMAN J. G. & DrVKns A. L. (1976) Isolation, characterizL~~~~HIxIx-L~F~HIR~ M. (1978) Predicted {i-turns in pepation and physical properties of protein antifreezes from tide and glycopeptide anti-freezes. Biockt,nt. hiopk~.\. R<,.s. the winter flounder. PsL,rtdop/ruro,l~,~r~,,s mwriamus. C‘o~wntrn. 81, 135’- 1356. cOF?lp. ~ioc’kt’rn. Ph,wird. 538, 375 -380. MA(.KI-NZI~ D. D. S.. MAACK T. & KINT~;K W. (1977) Dtsht~u J. G.. P~TT~.RSOX J. L.. KOZAX J. J. & Dr:V~rrs Renal excretion of Chlorophenol red and related organic A. L. (1980) Isoprestio determination of water binding hy acids in the intact flounder Ps~,~~riopic,uron~,~~~~.~ cmtilrritish antifrecle glycoproteins. Bioc,him hiopl~!,r. .dc.rcr 626. (‘LIIILIS. J. e\-p. zoo/. 199, 449~_457. 332 336. EASTMAX J. T.. DtiV~n:s A. L.. COAI.SON R. E.. NOKDQLIIST MOKKIS H. R.. THO&~PSONM. R.. OSLIC;A D. T.. AHMI,I) A. I.. C‘IIAM S. M.. VA’WtNNFtl>F: J. R. & FL.l.NkY R. E. R. E. & BOYI, R. B. (1979). Renal conservation of anti(1978) Antifrcczc glqcoproteins from the blood of an freeze peptide in atarctic eelpout. R~tj~~~~p~~j~~~ dtoctrhorrli. Antarctic fish. J. hitri. Clrrtir. 253. 5155-5162. h’uturc. L0tlil. 282. ‘717m2 IX. FLI:X.HIX N. H. ( 1970) f’i:c C/ielnico/ Pizwics of Ice. p. I I 1. MIILVIHII.I. D. M.. Gt:oc;~jtc;a~ K. F.. YFH Y.. DT;RI:%!ER Cambridge University Press. Camhridge. K., 0~11~;~ D. T.. WARD F. C. & FTF.NEY R. E. (1980) FLI:T(.H~:R G. L. t 1977) Circannual cycles of blood plasma Antifreeze glycoproteins from polar fish. J. hid. C/WV. freezing point and Na ’ and Cl concentrations in New255, 659 662. foundland winter flounder (P.\c’ltriopl~lr,.o,1(‘(.fi,s cl,trt*riNOR>~AU J. R. (1938) Coast fishes. Part 3, The Antarctic <~~tr~rr.st correlation with water temperature and photozone. ‘Discocrr~’ Rrp. 18, 1~105. period. Crrn. J. Zttof. 55, 789 795. O’GRAUY S. M.. CLARKE: A. & DFVRIES A. L. (1982a) Characterization of glycoprotein antifreeze biosynthesis FLI:T( HI:R G. L. (1981) Effects of temperature and photoperiod on the plasma freezing point depression: Cl in isolated hepatocytes from Puyothmiu horchgrroinki. J. concentration, and protein “antifreere” in Minter rzp. Zoo/. 220, 179.-l 89. O’GRADY S. M. & DEVRIES A. L. (1981) Osmotic and ionic lloundcr. c‘clt~. J. Zoo/. 59, 193 201.

640

ARTHCR L. DEVRI~~

regulation in polar fishes. J. esp. &fur. Bioi. ECU!. 57, 2 19-228. O’GRAUY S. M.. ELLORY J. C. & DEVRIES A. L. (1982b) Protein and glycoprotein antifreezes in the intestinal fluid of polar fishes. J. rsp. Biol. 98. 429-438. OLLA B. I... BEJIIA A. J. & MAKTIN D. (1975) Activity, movements. and feeding behavior of the cunner. i&f;uoltrhrctr ~dsnc~r~zc.s.and comparison of food habits with ., young tautog. ‘Ffurrtcq~cio&is. off Long Island. New York. ~rsirrrr &t/f. I(!. 73, 895-900. OSCC;A D. T. & FEENEY R. E. (1978) Antifreeze glycoprottrim from Arctic tish. J. hiclf. Chcr,~. 253, 53385343. OS~I~;A D. T.. WARO F. C.. YEH Y. 62 FEFNEY R. E. (1978) Cooperative functioning between antifreeze glycoproteins. J. hiol. Chrm. 2.53, 6669-6672. P~ULINC~ L. (19531 Gmcrctl Chen~i.srr.r. 2nd edn, pp. 344-347. W. H. Freeman, San Francisco. PALLING L.. Contv R. B. & BRAXON H. R. (1951) The structure of proteins: Two hydrogen-bonded helical configurations of the polypeptide chain. Proc. nritr~. .iicud. sci. I;.s.A. 37, 205-21 1. PIIKCY W. G. (1961) Season changes in osmotic pressure of flounder sera. Sciencv, 134, 193.. 194. P~TZI:L D. & D~VKII‘S A. L. (1979) Renal handling of peptide antifreeze in northern fishes. Bull. hft Desrrt Is/. hiol. Lrrh. 19, 17. 19. P~~ZEL D. & DEVKIES A. L. (1980) Renal handling of anionit and cationic antifreeze peptides in the glomerular wmter hounder. Buil. Afr De.srrr Isi. hiol. Luh. 20, 17 18. Prrzt~ D. & Rt;Vmns A. L. (1981) Functional lnorphoiogy of the flounder glomerular capillary wall Bull. Mt L)rsrrt 1~1. 5i0i. LUG. 21, 35-37. _ PETZ~L D.. R~ISMAN H. & D~VRI~S A. L. (1980) Seasonal variation of antifreeze peptide in the winter flounder. Pa~[tdoplrlfror~~[,r~~.sumerictmus. J. rrp. Zoo/. 21 1. 63369. RAYhfoND J. A. (1976) Adsorption inhibition as a mechanism of freezing resistance in polar fishes. PhD Thesis, University of California. San Diego, pp~ l-l 69. RA~YOXD J. A. & D~VRIES A. L. f 1972) Freezing behavior of fish blood glycoproteins with antifreeze properties. Cr~(~~j(~~~~~~ 9, 541-547. RAYMONU J. A. 6: DrVmrs A. L. (1977) Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Prr?c,. mtn. .4curl. Sc,i. L:.S.A. 74, 2589-2593. RA!‘hlOND J. A., LIN Y. & D~VRIES A. L. (1975) GlyCo-

proteins and protein antifreeze in two Alaskan fishes. J. cxn. Zaoi. 193, 125-130. RAYMOND J. A.. RADDING W. & DEVRIES A. L. 11977) Circular dichroism of protcin and glycoprotein fish antifreeze. Biopolrmrrs 16, 2.575.-2578. RING R. A. (1980) Insects and their cells. In Low Temprrucure ~ntl Prrserrarion in Medicine und Biolog_v (Edited by ASHWOOD-SMITH M. J. & FARRANT J.), pp. 187-217. Pitm;m Medical. Tunbridge Wells. SCHOLANIXZKP. F. & MAUXRT J. E. (1971) Supercooling and ice propagation in blood from arctic fish. Cr.&i+ io