X-Message-Number: 4732 Date: Mon, 7 Aug 1995 18:51:46 +0200 (MET DST) From: Eugen Leitl <> Subject: nature's cryonics (II) Temperature Adaptation cot'd (10 pages of 14). =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= as stated above, terrestrial invertebrates that tolerate ice formation in their extracellular fluids generally are in a state of months-long hibernation. Among aquatic animals the intertidal invertebrates of high latitudes may display impressive degrees of freezing tolerance. For instance, Kanwisher (1955) showed that two intertidal molluscs, Mytilus edulis, and Littorina rudis, withstand freezing of approximately 70 percent of their total body water at an ambient temperature -20 deg C, a common winter temperature for the North Atlantic intertidal regions where these specimens were obtained. All freezing was in the extracellu- lar space. Although the cells appeared shrunken and distorted under microscopic observation, they contained no ice crystals (Kanwischer, 1955). In a related study, Murphy and Pierce (1975) examined the ability of another intertidal mollusc, Modioulus demissus demissus, to acclimate to low temperatures and, thereby, to increase its tolerance of freeezing. They were able to demonstrate that a pronounced increase in the mollusc's capacity to withstand tissue desiccation accompanied low temperature acclimation. Specimens acclimated to 23 deg C died when 35 percent of the tissue water was removed via extracellular ice formation; 0 deg C-acclimated individuals could tolerate the loss of 41 percent of tis- sue water. An important conclusion from this study is that low tem- perature acclimation does not reduce the amount of tissue water lost during extracellular freezing, but instead leads to a greatly increased tolerance of partial dehydration. The molecular basis of this acclima- tory effect is not known, albeit the involvement of macromolecular antifreezes in certain intertidal molluscs (Theede et al., 1976) raises the possibility that these molecules may play some role in stabilizing bio- chemical structures and functions in face of reduced water activities, a suggestion that has also been made in the case of xerotolerant insects such as Tenebrio molitor (Patterson and Duman, 1979; Schneppenheim and Theede, 1980). /* Mike, whadda you say to this below: */ A final group of compound merits consideration in the context of freezing tolerance. These are polyhydroxyl alcohols (polyols) like glyc- erol, sorbitol, and mannitol, which may accumulate to high concentra- tions in many insects that are either freeze-tolerant or freeze-resistant. Polyols lower the supercooling point of a solution approximately twice as much as they reduce the true freezing point, so these compounds may be of great significance in the prevention of ice formation. The potential disadvantages of polyols are at least twofold. First, because they function in a strictly colligative manner, unlike the peptide and glycopeptide antifreezes discussed later, polyols will sharply increase the osmotic concentration of the body fluids. If polyols are uniformly Table 11-11. "Antifreeze" Proteins and Glycoproteins in Fishes and Invertebrates. -------------------------------------------------------------------------------------------- Group of organisms Type of Molecular weight Thermal hys- Reference "antifreeze" (daltons) teresis deg C -------------------------------------------------------------------------------------------- Antarctic fishes Glycoproteins 8 size classes 1.27 DeVries (1974) Notothenidae 00-33700 (e.g. Pagothenia borchgrevinkii) DeVries (1974) Zoarcidae Glycoproteins 0.76 (Rhigophila dearborni) Arctic fishes flounder Proteins 3 size classes 0.62 (winter) Duman and DeVries (Pseoudopleuronectes (1974, 1976) americanus) cod Glycoproteins 7 size classes 1.18 Van Voorhies et al. (Gadus ogac) same as Antarct. (1978) nototheniids saffron cod Glycoproteins 1.0 Raymond et al. (Eleginus gracilis) (1975) sculpin Protein 1.4 Raymond et al. (Myoxocephalus (1975) verruccosus) insects (Tenebrio molitor) Proteins several size Patterson and classes Duman (1979) Schnppenheim and Theede (1980) spider 2.44 (Feb.) Duman (1979) (Philodromus sp.) Proteins 0 (June) spider 1.88 (Jan.) Duman (1979) (Clubiona sp.) Proteins 0 (warm-accl.) beetle (Dendroides Proteins 3.62 (cold- Duman (1980) canadensis) accl.) beetle (Merancantha Proteins 3.71 (Feb.) Duman (1977) contracta) ------------------------------------------------------------------------------------------- distributed throughout all body fluid compartements, however, this osmotic effect may not be much of a problem,since redistribution of water among compartments would not occur. Second, the polyols in- crease the viscosity of body fluids greatly, a problem that again is avoided by the peptide and glycopeptide antifreezees, which work in noncolligative manners. One important feature of polyols like glycerol is that they may serve a variety of functions in organisms that experience freezing or desic- cating conditions. Glycerol functions as a cryoprotectant, as a super- cooling agent, and has been shown to aid in desiccation resistance in cysts of Artemia (Clegg, 1962). As noted in Chapter 10, glycerol is a compatible solute that lacks perturbing effects on proteins. Common inorganic ions, e.g. K+, Na+, and Cl-, do not exhibit compatibility at high ionic strengths. Thus, glycerol may be an appropriate solute to use at low water activities, whether these low activities are asso- ciated with high external osmotic concentrations, with encystment and dormancy, or with freezing tolerance. It is necessary to realize, how- ever, that glycerol is not an ubiquitous component of the body fluids of freeze-tolerant and freeze-resistant animals (Duman, 1980; Zacharias- sen, 1980). The full chemical armament of such animals usually con- tains other compounds whose contributions are critical for survival at low temperatures. These compounds are the peptide and glycopeptide antifreeze molecules, wwhich have now been described in a variety of high latitude fishes as well as in several terrestrial invertebrates. Freezing Resistance and Macromolecular Antifreezes. One of the most intriguing stories about molecular evolution to have been developed during the past two decades involves the macromolecular antifreezes (peptides and glycopeptides), first discovered in larvae of the insect, Tenebrio molitor (Ramsay, 1964) and blood sera of Antarctic fishes (reviewed by DeVries, 1980, 1982). The currently identified family of antifreezes listed in Table 11-11 reveals that these peptides and glycopeptides are found in a taxonomically diverse assemblage of ani- mals, differ substantially in chemical structure and molecular weight, and can vary in concentrations as a function of acclimation or acclima- tization. All of these antifreezes share one important common property, however, which is a diagnostic trait for those molecules: they lower the freezing point (temperature of ice crystal growth) of a solution more than the melting point (temperature of shrinkage of ice crystals). This "thermal hysteresis" is used to detect the presence of antifreezes in solutions and, moreover, may provide an important clue to the mech- anism of antifreeze action, as discussed later. We will see, in fact, that in spite of the diversity of antifreeze primary structures (Table 11-11), a common mechanism of antifreeze action is likely to apply in all cases. The glycopeptide and peptide antifreezes are common in high lati- tude fishes that inhabit the upper regions of the water column where ice is present (Table 11-11). Fishes of high latitude seas lacking these antifrezes must seek out ice-free niches, and many species are likely to remain in a supercooled state throughout their lives (DeVries, 1980). The antifreezes present in high latitude fishes are a fascinating example of convergent evolution at the molecular level. The evolutionary devel- opment of glycopeptides and peptides capable of causing thermal hysteresis has occured several times. Distantly related fishes have independently "discovered" the sane antifreeze primary structures (Fig. 11-27). The repeat tripeptide unit, "alanyl-alanyl-threonyl" (with a carbohydratemoiety attached tothe threonyl residue(, is found in the Antarctic nototheniid fishes, e.g, of the genus Trematomus and in the Arctic rock cod, Gadus ogac (Van Voorhis et al.l, 1978). The peptide antifreezes of the Arctic sculpin,Myoxocephalus verrucosus, and flounder, Pseudeopleuronectes americanus, likeise appear to have similiar structures. Cysteine-rich antifreezes have been described in both fish (the sea raven, Hemitrepterus americanus; Slaughter et al., 1981) and insects (Tenebrio molitor; Schneppenheim and Theede, 1980). Thus, there appears to be a considerable variety of molecular structures that can function as antifreezes, and the independant evolution of these different structures has occured again and again, in a variety of fishes and invertebrates. The structures of two types of antifreezes are shown in Figure 11-27. The glycopeptide antifreezes were the first to be sequenced, and they characteristically exhibit a simple structure in terms of amino acid sequence. Alanyl residues contribute a high percentage of total residues, and threonyl residues serve as the atachment sites for the carbohydrate components of the antifreezes. The basic repeated unit shown in Figure 11-27 is found in antifreezes of different molecular weights. The smallest glycopeptide is 00 daltons, and the largest is approximately 33000 daltons (DeVries, 1980). In the smalles class of glycopeptide, some of the alanyl residues are replaced by prolines. The partial sequence the peptide antifreeze from the winter floun- der, P. americanus, is shown in the lower panel of Figure 11-27. Alanyl residues again constitute a major fraction of the amino acid chain. Threonyl and aspartyl residues are the other dominant components of the peptide antifreezes. Like the galactose and galactose-amine residues of the glycopeptide antifreezes, the polar or charged threonyl and /* upper panel omitted due to the limitations of ASCII art */ Antarctic cod (D. mawsoni) ALA-ALA-THR-ALA-ALA-THR-ALA-ALA-THR-ALA- | | | Gal-A Gal-A Gal-A | | | Gal Gal Gal Winter flounder (P. americanus) ASP-THR-ALA-SER-ASP-ALA-ALA-ALA-ALA-ALA-ALA | _ | | | _ COO OH OH COO -LEU-THR-ALA-ASP-ALA-ALA-ALA-ALA-ALA-ALA- | | _ OH COO Fig. 11-27. Structures of the glycopeptide and peptide antifreezes of polar fishes. Upper panel: the basic repeating structural unit of a glycopeptide with antifreeze properties. The peptide chain contains only two types of residue (alanyl and threonyl). To each threonine residue is joined a disaccharide (beta-D-Galactopyranosyl-(1-3)-2-acetoamido-2-deoxy-alpha-D-galactopyranose. Lower panel: Primary structures of the glycopeptide antifreezes of the Antarctic nototheniid fish Dissostichus mawsoni (observe the ALA-ALA-THR- repeat unit, shown in the upper panel), and the peptide antifreeze of the winter flounder, Pseudopleuronectes americanus. Figure after DeVries (1980). aspartyl residues provide a "front" of polar groups which may facilitate strong adsorption to ice crystals, as discussed below. The insect antifreezes have not been as well characterized at the structural level. None of the insect antifreezes has been found to con- tain carbohydrate components, i.e. all of those antifreezes appear to be peptides (Duman et al., 1982). The percentage contribution of differ- ent amino acid residues differs form the fish antifreezes. The insect pep- tide antifreezes characteristically have low alanyl contents, for example. The antifreeze peptide from the milkweed bug, Oncopeltus fasciatus, has a high serine content (30.5 percent; Patterson et al., 1981). Other insect antifreeze peptides contain high cysteine contents and lose their thermal hysteresis properties when treated with reagents that reduce disulfide bridges (Duman et al., 1982). How the secondary structure established by -S-S- bridges contributes to antifreeze functions is not presently known. Mechanisms of Antifreze Action. How do these diverse glycopep- tide and peptide antifreezes establish the thermal hysteresis which is their diagnostic characteristic? Ho can these antifreezes function in a noncolligative manner, i.e., how are they able to reduce the freezing point (temperature of ice crystal growth) of a solution more than can be explained on the grounds of total number of antifreeze particles in solution? One clue to the mode of function of these antifreezes is the high concentration of polar or charged groups along the antifreeze primary structure (Fig. 11-27). In the glycopeptide antifreezes, the sugars provide a regular front of -OH groups, since all of the hydrox- yls are thought to orient in one plane along the linear, expanded anti- freeze molecule (DeVries, 1980). In the peptide antifreezes, which have high percentages of threonyl and aspartyl residues, a similiar polar front is created. These polar groups appear appropriately spaced to facilitate a strong interaction with ice (DeVries, 1980). Thus, the antifreezes have been proposed to exert their effect by strongly absorbing to ice and making the addition more water molecules to the growing ice front less thermodynamically favorable (a more detailed discussion of this "poisoning" of growth mechanism is given by Raymond and DeVries, 1977). Evidence in support of this hypothesis has come from several types of experiments. For example, unlike solutes that obey colligative rela- tionships, the antifreezes do not freeze out of a solution as ice forma- tion occurs (Duman and DeVries, 1973). This finding suggests that a strong interaction takes place between the ice and the antifreeze. Using scanning electronmicroscopy, Raymond and DeVries (1977) provided striking visual proof of this interaction. They showed that the anti- freezes did adsorb to ice crystals, inhibiting their growth and affecting the crystal form of the ice that propagate. The thermal hysteresis observed in the freezing-melting behaviour of solutions containing anti- freezes further suggests an unusual interaction between ice and these molecules. The reduced freezing point observed in antifreeze-contain- ing solutions may reflect the inhibition of ice crystal growth that results when antifreeze molecules adsorb to the growing front of a small ice crystal. Once ice is formed, however, its melting temperature is that of normal ice. The dependance of antifreeze action on polar groups has been demonstrated by chemical modification studies, e.g., by blocking the hydroxyl groups on the galactose residues with sodium borohy- dride (DeVries, 1980). The removal of these polar groups may prevent efficient hydrogen bonding between antifreeze and ice, thereby elimina- ting the molecules' capacities to bind to ice and to inhibit further crystal growth. It is important to point out that the unusual capacities for interac- tion with ice posessed by the peptide and glycopeptide antifreezes do not mean that these antifreezes have atypical interactions with liquid water (see DeVries, 1980). The quantity of water bound to the anti- freezes is similar to the amount bound by proteins of similiar size which lack the thermal hysteresis properties of the antifreeze molecules. The fraction of serum water that could be bound by th antifreezes is only about 1 percent (DeVries, 1980), and this minute amount of bound water cannot account significantly for the freezing point depressing abilities of the glycopeptide and peptide antifreezes. What is the Relationship Between in Vitro and in Situ Function of Antifreezes? The elucidation of the mechanism of antifreeze action has, of course, involved in vitro experimentation. The careful studies of crystal growth in vitro may mislead us into thinking that a similiar phenomenon occurs throughout the body fluids of antifreeze- containing animals. In reality, ice formation per se must be avoided in most freeze-resistant species, notably the Arctic fishes which live continuously at -1.86 deg C (DeVries, 1980). Thus, even if the antifreezes in these fishes were capable of preventing the growth of ice crystals in the blood or cytosol, there is apparently no way to remove these crystals once they are formed. That is, the fish never reach temperatures as high as the melting point of the antifreeze-containing body fluids (approximately -1 deg C). These considerations have led ot the hypothesis that the in situ func- tion of antifreezes is one of inhibiting ice crystal propagation across integumental surfaces (DeVries, 1980). For example, in fishes the most likely site of ice propagation is across the gills, whih lack a covering of protective scales and mucus. Antifreeze molecules in the gill mem- branes and circulating fluids may, therefore, be critical in preventing the passage of minute ice crystals from the seawater into the body fluids. Schneppenheim and Theede (1979) have observed that the integ- ument of the Arctic sculpin, Myoxocephalus scorpius, contains anti- freeze peptides, a finding in support of the hypothesis that one of, if not the key, function(s) of the antifreezes is a "peripheral defense" involving the blocking of the ice crystal penetration into the interior of the organism. Antifreezes, Nucleating Agents, and Polyols in Insects: What are Their Roles? Unlike polar fishes in which each species contains =-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-=-= Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=4732