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Metabolic adaptation to temperature in eurythermal species may involve a change in the quantity or concentration of enzyme, as observed above in Sidell et al.’s goldfishes, or to actually change the enzyme to one that functions better at the new temperature. For those species that are genetically equipped to do so, the change in enzyme may be the result of an enzyme produced by a different gene locus (a different isozyme) or by a different allele of the same gene locus (a different allozyme).

For example, rainbow trout, Oncorhynchus mykiss, shows multiple isozymes of acetyl cholinesterase that can function optimally at different temperatures. The effect of temperature on their apparent Michaelis constant (K_m) is shown in Figure 2.9. The Michaelis constant is the substrate concentration at which the reaction proceeds at 50% of its maximum velocity (V_max). The K_m of isozymes in trout acclimated to cold (2 °C) differs considerably from that of trout acclimated to 18 °C (Baldwin and Hochachka 1970; Baldwin 1971). The K_m of the cold‐acclimated trout stays within a fairly flat functional range of 0.2–0.5 mM between temperatures of 0 and 14 °C, then above that range the K_m shoots way up. Similarly, the K_m for the warm‐acclimated trout at a temperature below 14 °C is very high, then drops to within the functional range between temperatures of 14 and 24 °C, and then shoots up again. Enzymes from the cold‐ and warm‐acclimated trout show different electrophoretic mobilities, meaning that the proteins are structurally different. Thus, within the trout at least, it is possible to have two different versions of the same enzyme, each of which functions optimally only over part of the seasonal temperature range encountered by the species. In practice, such a multiple isozyme solution to temperature change is a pretty rare occurrence. Trout are tetraploid, having four copies of each chromosome, instead of the diploid structure that we, and most oceanic fauna, have. The trout’s larger genome allows for more flexibility in dealing with environmental challenges (cf. Somero 1975; Hochachka and Somero 1984).

Figure 2.8 Changes in enzyme activity as a function of time after changes in acclimation temperature. Cytochrome oxidase activity (mean ± SE) of goldfish muscle homogenates from fish which were transferred from 15 to 5 °C; Cytochrome oxidase activity (mean ± SE) of goldfish muscle homogenates from fish which were transferred from 15 to 25 °C. Note that values are expressed per milligram protein.

Source: Sidell et al. (1973), figures 1 and 2 (p. 210). Reproduced with the permission of Springer‐Verlag.

Figure 2.9 The effect of temperature on the K_m of acetylcholine for acetylcholinesterase enzymes of four species of fish acclimated to different environmentally relevant temperatures: rainbow trout, acclimated at 2 and 18 °C; Pagothenia borchgrevinki, an Antarctic species; and the mullet (Mugil cephalus) and Ladyfish (Elops hawaiensis), two tropical species. The approximate temperature to which each fish is adapted or acclimated is given in parentheses below the fish's name.

Source: Hochachka and Somero (1973), figure 7‐14 (p. 231). Reproduced with the permission of Saunders Publishing.

Diploid species have two copies of each gene, the two alleles, of which one is normally dominant. A study by Place and Powers (1979) observed the expression of two allozymes for LDH B in the killifish Fundulus heteroclitus. LDH B is the type normally found in the heart, as opposed to LDH A normally found in skeletal muscle. Fundulus is a widely distributed species along the east coast of the USA, and the Place and Powers study used specimens obtained from Maine to Florida. They found that the two types of LDH B had different efficiencies with respect to temperature, one more efficient in the cold and the other more efficient at warm temperatures. The presence of the different allozymes scaled with the environmental cline in temperature along the eastern US seaboard.

It should be noted that few species have as wide a temperature tolerance as does Fundulus or as the brown bullhead mentioned earlier in this chapter. The great majority of species, particularly those in the open ocean, have a more restricted temperature range, and therefore geographic range, over which they are found. The question then remains as to how much temperature change warrants a change in enzyme type (and perhaps species!) in a more typical situation.

A good example of a more typical situation is provided in a study by Graves and Somero (1982) of Pacific barracudas, congeneric pelagic fishes with similar ecologies differing in habitat temperature by only a few degrees: 6–8 °C. The four congeneric species were Sphyraena argentea and S. idiastes, both cold‐temperate species, S. lucasana, a warm‐temperate species, and S. ensis, a tropical‐subtropical species. The study used purified enzymes of LDH A to evaluate the effect of temperature on the K_m (Michaelis constant) and the K_cat of the four species. K_cat is the catalytic efficiency of an enzyme, specifically the rate at which substrate is converted to product per unit time, per active site. Thus, activity of an enzyme = (K_cat) × the concentration of the enzyme.

The electrophoretic properties of the four species were separated into three patterns, with the two cold‐temperate species (S. argentea and S. idiastes) showing identical mobility. S. lucasana and S. ensis (T‐ST) were different from one another, and both were different from S. argentea and S. idiastes. The electrophoretic study suggested three different enzyme structures, one for the two cold‐temperate species, and one each for the warm‐temperate and tropical‐subtropical species. A look at the kinetic characteristics of the enzymes showed that they differed in those properties as well.

Figure 2.10 is a plot of K_m vs. temperature for the LDHs of three of the four species. Since the plots for the two cold‐temperate species, S. argentea and S. idiastes, were identical, only S. argentea is shown. The enzymes from the three species clearly show differences in their K_m vs. temperature curves, indicating that the enzyme has changed in character among the three locations. When the data are examined in more detail (Table 2.1), it can be seen that the K_m and K_cat at the temperature mid‐range for each species are virtually identical, but the enzymes show substantial differences when examined at a common temperature. The implications of the data are, first, that a change of <5 °C in a species’ temperature range can result in a change in the biochemical makeup of closely related species, and second, that within the barracudas studied here, K_m and K_cat both show highly consistent values at environmental temperature.

Figure 2.10 The effect of temperature on apparent Michaelis constant (K_m) of pyruvate for the M₄‐LDH's of three eastern Pacific barracudas. The K_m values for the southern temperate barracuda, S. idiastes (not shown), are statistically indistinguishable from those of the northern temperate species S. argentea. Error bars represent standard deviations. Each point represents an average of K_m values determined with at least three different purified LDH preparations from different individuals of each species. Solid portions of the lines connecting the K_m values indicate habitat temperature ranges of the species.

Source: Graves and Somero (1982), figure 3 (p. 103). Reproduced with the permission of John Wiley & Sons, Inc.

Table 2.1 K_m and K_cat values of pyruvate for three congeneric species of barracudas (genus Sphyraena) from different habitat temperatures at 25 °C and the temperature mid‐range (TM) for each species. S. argentea is a cold‐temperate species, S. lucasana is a warm‐temperate species, and S. ensis is a tropical‐subtropical species.

Source: Graves and Somero (1982), table 5 (p. 104). Reproduced with the permission of John Wiley & Sons, Inc.

	S. argentea	S. lucasana	S. ensis
Km of pyruvate at 25 °C	0.34 ± 0.03 mM	0.26 ± 0.02 mM	0.20 ± 0.02 mM
Kcat at 25 °C	893 ± 54 s−1	730 ± 37 s−1	658 ± 19 s−1
Temp. midrange (TM)	18 °C	23 °C	26 °C
Km of pyruvate at TM	0.24 mM	0.24 mM	0.23 mM
Kcat at TM	667 s−1	682 s−1	700 s−1