A M . ZOOLOGIST, 11:159-167 (1971).
Biochemical Adaptation to the Environment
GEORGE N. SOMERO AND PETER W. HOCHACHKA
Department of Zoology, University of British Columbia,
Vancouver 8, Canada
SYNOPSIS. Biochemical adaptation to environmental parameters such as temperature
appears to involve two distinct types o£ changes in the organism's chemistry. On the
one hand, the quantities of certain molecular species present in the cells may change.
Alternatively, the actual types of molecules present may vary. Rainbow trout (Salmo
gmrdnen) acclimated to warm and cold temperatures exhibit a striking example of
this latter type of adaptation. For all enzymes we have examined in this species,
distinct "warm" and "cold" isozymes are present. The isozymes found in warmacclimated (18°C) trout function well only at temperatures above 10-12"C. The
isozymes present in cold-acclimated (4°C) trout function optimally at 2-5°C, temperatures this species normally encounters in winter. These data, plus information on
comparable changes in membrane lipids, lead us to propose that adult poikilotherms
may undergo a considerable degree of "biochemical restructuring" on a seasonal basis.
The factors which control this "restructuring," and the rates at which the process
occurs at high and low temperatures, are topics for future investigation.
Although we shall iocus our attention
almost entirely on adaptation to a single
physical parameter, temperature, it would
seem useful to preface this essay with a
brief discussion of a well understood biochemical adaptation to the chemical environment. Perhaps the classical example of
environmental adaptation, at least in the
opinion of biochemists, is the adaptation
of micro-organisms to changes in the nutrient content of their environment. For example, the addition of a simple sugar to
the culture medium in which a microorganism has been growing will soon result
in the induction of sets of enzymes necessary for the transport of this sugar into the
cells and for the subsequent utilization of
the sugar in energy metabolism. We cite
this example in order to lay a firm foundation for what we feel might be termed a
"paradigm of biochemical adaptation to
the environment." That is, biochemical
adaptation to the environment is effected
by producing the proper molecules in the
proper quantities at the proper time to
ensure satisfactory biological function in
the face of new environmental conditions.
We shall show that much as bacteria are
capable of producing the right enzymes to
Present Address of George N. Somero: Scripps
Institution of Oceanography, University of California, San Diego, La jolla, California 92037.
take advantage of changes in their nutritive environment, so can cold-blooded
(poikilothermic) organisms produce the
necessary enzymes to enable the organisms'
metabolic functions to continue under
quite extreme conditions of thermal stress.
Before we begin a detailed examination
of temperature adaptation in poikilotherms, it is essential to emphasize that the
mechanisms we shall discuss may be characteristic only of those poikilotherms
which are incapable of avoiding thermal
stress through such avenues of escape as
partial heterothermy and behavioral regulation of temperature. That such escape
mechanisms do exist in numerous forms
among poikilotherms shoud be abundantly
clear from the papers of others in this
symposium (e.g., Carey and Teal; Heath
et al.).
The types of thermal stress encountered
by true poikilotherms can be conveniently
grouped into three classes on the basis of
the time-course of the stress. At one extreme is evolutionary adaptation to temperature, a process by which a species
adapts to a new thermal regime over many
generations. The end result of this long
term process is a high degree of similarity
in the rates of physiological processes
among organisms from widely different
157
158
GEORGE N. SOMERO AND PETER W.
Temperature (°C)
FIG. 1. In vitro oxygen consumption levels (Qoa)
of Trematomus gill filaments, expressed as ^1
O» consiimed/mg dry wt tissue/hr. From Somero
et al. (1968) and Ekberg (1958).
latitudes (Scholander et al., 1953; Wohlschlag, 1964; Somero et al, 1968). An
excellent example of evolutionary cold
adaptation is found in the Antarctic Nototheniid fishes such as Trematomus bernacchii. The whole organism and tissue
respiratory rates exhibited by this species
at its normal habitat temperature of
— 1.9°C are roughly the same as the rates
characteristic of temperate zone species at
their much higher habitat temperatures
(Fig- !)•
The second time-course of thermal stress
to be discussed is also illustrated in Figure
1. Much as different species may exhibit
similar rates of physiological activity in
spite of widely different habitat (body)
temperatures, so may an individual of a
single species show compensatory adjustments in physiological rates on a seasonal
basis. This process, which occurs during
the life span of the individual organism, is
commonly termed "acclimitization," if the
changes occur in the organism's natural
habitat, or "acclimation," if the changes
occur in the laboratory and in response to
variation in a single parameter, e.g., temperature. At this point it seems pertinent
to raise the following question: Is the similarity in the physiological end-results of
the evolutionary adaptation process and
the acclimitization (or acclimation) process due to an identical biochemical basis
for these two temporally disparate processes? We shall indicate later that at least
a partially affirmathe answer can be given
to this question.
HOCHACHKA
Finally, at the other extreme of the timecourse scale, one observes thermal
stresses which occur on a diurnal basis.
Stresses of this frequency are particularly
acute in the case of intertidal organisms
which may encounter 10-20°C changes in
body temperature over periods of a few
hours. Quite surprisingly, at least on the
basis of the dogma of classical physiology,
many intertidal organisms have been
shown to exhibit essentially temperatureindependent rates of physiological activity
(Newell, 1966; Newell and Northcroft,
1967; Baldwin, 1968; Newell and Pye,
197Qa,b). What biochemical mechanisms
can possibly permit Q10 values approximating unity?
In this essay we shall attempt to summarize the known information on the biochemical mechanisms by which physiological processes are maintained at relatively
temperature-independent rates, regardless
of the time-course of thermal stress. Our
summary of the existing data should serve
not only to illustrate what is known, but
also to point out what gaps remain in our
knowledge and what avenues of attack on
the remaining questions would seem most
fruitful.
If we equate metabolic processes with
enzymic reaction sequences, then we can
readily list the basic types of biochemical
changes which could possibly serve to elevate metabolic processes at low temperatures. First, and perhaps most simply, the
quantities of enzymes present in the cell
may increase during cold adaptation.
Changes in the concentrations of ratelimiting enzymes would seem of particular
importance. Quite surprisingly, in spite of
the inherent appeal of this hypothesis, no
unequivocal measurements of enzyme concentrations in warm- and cold-adapted organisms have been made. Indeed, there are
many studies in which the gross activities
of enzymes in tissues from warm- and coldacclimated organisms have been estimated
(e.g., Hochachka and Somero, 1971). In
no case, however, have changes in enzymic
activity been shown to be due to actual
changes in en/yme concentration. Thus,
verification of this hypothesis remains a
159
BIOMEDICAL ADAPTATION
major area for future study.
A second important manner in which
enzymic activity can be altered involves
changes in the intracellular environment
in which the enzymes function. One can
list a large number of intracellular changes
which can affect enzymic activity: (1) alterations in the concentrations of enzyme
activators and inhibitors; (2) changes in
intracellular pH; (3) changes in the membranes to which enzymes are bound; (4)
changes in substrate concentrations; and
(5) changes in hormone levels. The occurrence of such changes in temperature
adaptation is incompletely charted. It is
known that major alterations occur in
membrane phospholipids (Johnston and
Roots, 1964; Roots, 1968; Anderson, 1970)
and that such alterations can drastically
modify the properties of lipoprotein transport systems in the membrane (Wilson et
al., 1970). Changes of this nature may be
particularly important in adaptation of the
nervous system. Intracellular pH also
varies with temperature due to the temperature-dependence of the pK of water
(Rahn, 1965); pH changes could therefore
be of importance in all time-courses of
adaptation. The concentration of tissue
and serum ions may also change dramatically during acclimation (Hickman el al.,
1964). These kinds of modification of intracellular microenvironment can lead to
important metabolic adjustments and can
provide at least a partial answer to our
question about the biochemical basis of
temperature adaptation.
Finally, there is a third important way
in which enzymic activity may be regulated
for thermally-independent function. Adaptation to temperature may involve the
production of enzymes which are particularly well suited for function at the
temperature to which the organism is
adapted. Thus, an enzyme from a coldadapted organism might be a better catalyst at low temperatures than the homologous form of the enzyme found in a warmadapted organism. To appreciate how
the catalytic function of an enzyme might
be improved, it is helpful to consider a
simplified version of an enzymic reaction
Vma:
[SUBSTRATE] x IO4M
FIG. 2. The effect of varying enzyme-substrate
affinity, defined as the reciprocal of the Michaelis
constant (Km), on enzymic activity. Enzymes A
and B catalyze the same reaction and exhibit the
same activity at saturating (Vmax) concentrations
of substrate. The enzymes have a two-fold difference in Km. At physiological substrate concentrations (indicated by stippling) enzyme A is much
more active than 15. In terms of enzyme regulation
theory, B may represent a deactivated state o£ the
enzyme and A may represent an activated state. B
may be converted to A by the binding of a positive
modulator; conversely, A may be converted to
B under the influence of a negative modulator.
sequence, symbolized: E -\- S = ES = E -fP. The first step, or equilibrium, in this reaction involves the reversible formation of
an enzyme-substrate complex (ES) from
free enzyme (E) and free substrate (S).
To cause an improvement in function, one
would predict that the propensity for ES
complex formation could be increased. As
an approximation of this propensity for ES
complex formation, biochemists customarily refer to the "apparent enzyme-substrate
affinity," which is often equated with the
reciprocal of the apparent Michaelis constant (Knl) of substrate. This latter parameter is the concentration of substrate necessary to yield half the maximal velocity of
the reaction; when the Km (determined by
kinetic studies) is identical with the binding constant (determined by binding tests),
it is a good measure of enzyme substrate
affinity. Intuitively, it seems clear that the
smaller the amount of substrate necessary
to half-saturate the enzyme, the higher the
affinity between enzyme and substrate molecules (Fig. 2). It is known, in fact, that ES
affinity is a vitally important factor in the
modulation of enzyme activity in the cell
(Atkinson, 1969). Most positive modulators (activators) of enzymes exert their
influence by increasing ES affinity (lowering the apparent Michaelis constant of
160
GEORCE N. SOMERO AND PETER W. HOCHACHKA
through temperature-dependent changes
in ES affinity may obviously be of major
importance to some organisms, such as
those in intertidal zones, which encounter
large diurnal changes in habitat (body)
temperature.
Although the apparent Km usually exhibits a direct relationship to temperature
over the biological temperature range, we
have found that sharp increases in this
parameter often occur at temperatures at
or below the organism's normal temperature range. For example, the catalysis of
pyruvate kinase of rainbow trout acclimated to temperatures of 10-17°C (Fig. 4)
would appear to be severely limited at
temperatures much below 10°C. This enzyme certainly would be largely incapable
of binding substrate, under conditions of
0
5
10
15
physiological substrate concentration, at
TEMPERATURE (°C)
temperatures normally experienced by AntFIG. 3. The effect of temperature on the Mi- arctic fishes. In addition, one can see that
chaelis constant of glucose-6-phosphate (G6P) of
the summer trout would also have greatly
king crab leg muscle G6P dehydrogenase. (From
reduced
pyruvate kinase function at this
Somero, 1969).
species' winter temperatures of 2-4°C. The
substrate); negative modulators usually fact that trout remain active over a temhave the opposite effect. With these consid- perature range in which an enzyme such as
erations in mind, it became clear that the pyruvate kinase (Fig. 4) would function
effect of temperature on ES affinity could poorly led us to examine enzymes from
be a most important factor in thermal
adaptation. This expectation has been fully realized.
As illustrated in Figure 3, ES affinity
normally increases as the temperature decreases over the species' biological temperature range. In oilier words, the following
important analogy is seen to hold: Over
the biological temperature range, temperature decreases act analogously to positive
modulators of the enzyme. The biologically important result of this relationship between temperature and ES affinity is that,
at the low substrate concentrations normally found in vivo (roughly 10~4 M and
below), rates of enzymic activity may be
largely independent of temperature. It is
important to stress that, were substrate
10
15
20
concentrations in the cell not below saturTEMPERATURE (JC)
ating levels, Knl changes, whether in response to modulators or to temperature FIG. 4. The effect of temperature on the Miconstant of phosphoenolp\ru\ate (PEP) for
changes, could exert no influence on rates chaelis
several puuvate kinase>>. (Data from Somero and
of en/vmic function. Reduction in O10 Hochachka, 1968, and Somero, 1969).
161
BIOMEDICAL ADAPTATION
0
20
25
Temperature, °C
FIG. 5. The influence of temperature on the Km
values of phosphoenolpyruvate (PEP) for pyruvate kinases of 2° and 18°C acclimated trout.
Adult rainbow trout weighing approximately 175 g
were acclimated for 4-5 weeks. Specimens were fed
ad libitum dining holding. To prepare the enzymes
white epaxial muscle was homogenized with
4-5 vols. of 0.01 M tris/HCl buffer, pH 7.4, containing 1 mM EDTA. The crude homogenate was
centrifuged for 20 minutes at 12,000 g and the
pellet discarded. The supernatant was brought to
40% saturation with solid ammonium sulfate. The
suspension was centrifuged as above and the resulting supernatant was brought to 75% saturation
with ammonium sulfate. The precipitate was collected and redissolved in 0.01 M tris/HCl buffer,
pH 7.4. Dialyzed aliquots of this preparation were
used in the assays. Enzyme activities were assayed
spectrophotometrically. Km values were determined
by double-reciprocal
(I/velocity versus 1/S)
plots. At all temperatures pH values were held
constant.
cold-acclimated specimens. For all enzyme
systems examined (Figs. 5, 6, 7, 8, 9), distinct "summer" and "winter" variants of
the enzymes were present. In each case,
acclimation to low temperature led to the
appearance of a new enzyme variant which
exhibited a minimal Km value at temperatures 5-10°C lower than the minimal Km
temperatures observed for the "summer"
variants.
The importance of these changes in the
seasonal acclimation process can best be
appreciated by considering how a "summer" enzyme variant would function at the
trout's winter temperatures. The first
disadvantage of the "summer" variant is
5
10
15
Temp t C I
20
!5
FIG. 6. Effect of temperature on the Km of isocitrate for soluble NADP-isocitrate dehydrogenase
from liver of rainbow trout acclimated to 4° and
to 17°C. The enzymes were partially purified by
ammonium sulfate fractionation. The Km of
NADP varies with temperature in a similar manner. (From Moon, 1970).
20
25
Temperature, °C
FIG. 7. The influence of temperature on S0.5 values of fructose-6-phosphate (F6P) for muscle phosphofructokinases from 2° and 18°C acclimated
trout. The enzyme was prepared as described for
PyK except that the pH of the solutions was
maintained at 8.5, and the protein fraction used for
the assays precipitated between 35 and 50%
ammonium sulfate saturation. The spectrophotometric assay mixture included 0.05 M tris/HCl
buffer, pH 8.5; 0.10 mM ATP; 1.0 mM MgCU; 30
jig of Sigma a-glycerophosphate dehydrogenasetriosephosphate isomerase; 120 fig of Sigma rabbit
muscle aldolase; varying concentrations of F6P: and
PFK, added last. S0B values were determined from
Hill plots.
162
GEORGE N. SOMERO AND PETER W. HOCHACHKA
present. Clearly, the maintenance of relatively stable rates of enzymic function in
the face of sudden changes in body temTABLE 1.
The influence of temperature on the
pyruvate Icinase reactions catalyzed by the "summer" and "winter" variants at different substrate
concentrations.
Concentration of
substrate (PEP) V7VV=°
'Summer" Pyk
-
Temperature f'C)
FIG. 8. Effect of temperature on the Km of
brain acetylcholinesterase of Trematomus (open
circles) 2°C acclimated rainbow trout (solid
squares), 17°C acclimated rainbow trout (dark
circles) and of the electric eel (solid triangles).
The enzymes from Trematomus and trout were
partially purified by methods described in Baldwin
and Hochachka (1970). The enzyme of electric eel
was a purified preparation obtained from Sigma
Chemical Company. (Data from Baldwin and Hochachka, 1970, and Baldwin, 1970).
apparent: The rate of catalysis per se is
lower than in the case of the "winter"
variant. Second, it should be noted that
the "summer" variant also has a limited
ability to vary its rate of catalysis as the
concentration of substrate varies in the
cell. Thus, at low temperatures the "summer" enzyme is poorly equipped to regulate its activity in response to changes in
substrate levels which might occur during
vigorous metabolism.
A third disadvantage of the "summer"
variants at low temperatures stems from
the fact that, over the winter temperature
range (generally below 10°C), decreases
in temperature promote decreases in enzyme-substrate affinity. Consequently, the
temperature-dependence of the reactions
catalyzed by the "summer" enzymes is extremely large. For example, consider the
temperature-dependence of the "summer"
and "winter" pyruvate kinase reactions at
low temperature and at low substrate concentrations (Table 1). At winter temperatures, variations in habitat (body) temperature would be accompanied by changes in
pyruvate kinase activity characterized by
Q 10 \alues approximating 20 if only the
"summer" variant of the enzyme were
'Winter" Pyk
1
1
5
1
1
5
3
X 10" M
X 10-'M
X 10-=M
X 10"3M
X 10"*M
X 10"5M
2.5
3.2
3.6
1.4
1.6
1.7
Q108-7°°
19.8
24.6
2.2
3.5
3.6
r
FIG. 9. Effect of temperature on the Michaelis
constant of oxaloacetate and acetyl Co A on liver
citrate synthases, which were partially purified
from trout acclimated to 2"C and 18°C. At temperatures above 9°C, estimates of Km were highly
reproducible for both enz)me preparations. For
example, in the case of the 2° enzyme, three
independent estimates of the Km for oxaloacetate
at 22°C, pH 7.5, yielded 0.022 mM in each case.
The Km estimates at temperatures below 9°C were
from dilferent en/\me preparations. (From Horharhka and Lewis, 1970).
BIOMEDICAL ADAPTATION
163
TABLE 2. Biochemical changes associated with temperature acclimation in poikilothermic organisms. See
Hochaclika and Somero (1971) and Precht (1968) for literature dealing with these topics.
Constituent
Enzymes
Lipids
Metabolic pathways
Protein synthesis
Nucleic acid synthesis
Blood ions
Tissue ions
Bibosomes
Hemoglobins
Change
Different variants in winter and summer rainbow trout
Changes in saturation, chain length and quantity of lipid
Increases in activities of pathways associated with biosynthesis in cold-acclimated
fish, e.g., increase in hexose monophosphate shunt
Change in rate
Change in rate
Changes in relative concentrations
Same as above
Differences in ribosome melting temperatures between summer and winter trout
Changes in O2 affinity likely due to changes in modulator concentrations
perature demands that "winter" forms of data which suggest that enzymes from
the enzyme be present.
poikilothermic forms have a higher catTo conclude this section on enzymic alytic efficiency, in terms of the substrate
adaptation to temperature two points turnover number, than do enzymes from
should be stressed. First, it seems clear that homeothermic species (Assaf and Graves,
evolutionary adaptation and seasonal ac- 1969). Comparisons of "winter" and "sumclimitization (or acclimation) are promoted mer" enzyme variants would obviously be
by a common biochemical mechanism, of great interest.
namely the production of enzyme variants
At the beginning of this essay we formuwhich exhibit minimal Knl values at tem- lated a "paradigm of biochemical adaptaperatures approximating the lowest tem- tion to the environment," which stated
peratures experienced by the organisms in that adaptation involves the production of
their habitats. Second, it is important to the proper molecules in the proper
point out the limits to which enzyme-sub- amounts to ensure successful biological
strate affinity changes can promote rate com- function under new environmental condipensation. For the majority of enzymes we tions. In all the enzyme systems we have
have examined from winter and summer examined in trout, changes of this sort
rainbow trout, the minimal Km values of have been observed. To indicate that qualthe two seasonal variants are approximate- itative changes are not restricted to enly equal. Thus, in the absence of changes zymes alone, we have summarized in Table
in enzyme concentration on a seasonal ba- 2 the other types of biochemical changes
sis, there would still be quite large differ- which occur during temperature acclimaences in total enzymic activity between tion. It is clear that the acclimation process
winter and summer specimens. For en- is characterized by a major biochemical
zyme-substrate affinity changes to promote restructuring of the organism. At the
complete temperature compensation, the present moment the adaptive significance
"winter" variant of the enzyme would have of certain of these changes is not clear. For
to exhibit higher absolute affinity for sub- example, how do "summer" and "winter"
strate than the "summer" variant. This ribosomes differ in their protein synthetic
type of difference does not appear to be the abilities? How do altered phospholipids
rule.
affect the activities of membrane-bound
To promote rate compensation to tem- enzymes? However, we feel that when anperature there may be a second important swers to these questions are sought, it will
way in which an enzyme can be "im- again be found that these other biochemiproved" for low temperature function: cal restructuring changes will be adaptive,
The turnover number per se may be much as we know that changes in enzyme
greater for a cold-adapted enzyme than for variants are.
a warm-adapted enzyme. Tests of this hyTo conclude this essay, we now would
pothesis are very limited, albeit there are like to briefly consider some of the major
164
GEORGE N. SOMERO ANO PETER W.
Change in temperature. . Endocrine or
neural response
(orphotoperiod??)
HOCHACHKA
. Increased protein and. . New ribosomes - . New steady
state
nucleic acid synthesis
new isozymes
Transitory Changes
1. Altered rates of protein and nucleic acid
synthesis.
2. Activation of metabolic pathways associated with biosyntheses.
New Steady State
New isozymes.
New ribosomes.
New membrane lipids.
Altered ionic compositions of body fluids
and tissues.
5. Altered metabolite pools.
1.
2.
3.
4.
FIG. 10. Hypothetical acclimation scheme
unanswered questions concerning temperature adaptation. Clearly, we currently have
a fair idea about what is occurring to
promote temperature adaptation. The important questions remaining would seem
to concern: (1) how are these changes
brought about, and (2) how do these
changes occur sequentially?
In Figure 10 we propose a hypothetical
scheme for the time course of the acclimation process. Indicated in this scheme are
several of the uncertainties remaining to
be resolved. For example, the nature of the
primary inducing stimulus is still uncertain. It is possible that photoperiod and
temperature interact to induce the acclimation changes. We have noted that coldacclimation
in summer and warmacclimation in winter are extremely difficult to effect.
Once the fact of environmental change
is perceived by the organism, then it must
respond by initiating the biochemical
changes characteristic of acclimation. What
mediates this response? Are neural stimuli
important? Are hormone messengers involved? Is a direct effect of temperature on
the cells enough to promote acclimatory
changes in metazoan organisms?
After the biochemical changes are initiated, then one must ascertain which
changes are truly characteristic of the final
steady state and which changes merely
serve to bring about the final state. For
example, are the increased rates of protein
and nucleic acid synthesis noted during
cold-acclimation likely to be maintained
after the new isozymes, new lipids, etc.
have been synthesized? Might, for example, changes in the activity of metabolic
pathways associated with biosynthesis, such
as the hexose monophosphate shunt, be
transitory events which will be observed
only during the time before a new steady
state is attained? If certain changes are
merely effectors of the final steady state,
then might not these transitory effects be
characteristic of both warm and cold acclimation? Clearly, a critical examination of
the factors inducing acclimation changes
and of the time-course of the process itself
is due.
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