Effects of temperature acclimation on lactate dehydrogenase of cod (Gadus morhua): genetic, kinetic and thermodynamic aspects
1 Department of Biology, University of Antwerp, Groenenborgerlaan 171,
B-2020 Antwerp, Belgium
2 Department of Fisheries and Marine Biology, University of Bergen, PO Box
7800, N-5020 Bergen, Norway
3 Marine Biology/Ecological Physiology, Alfred-Wegener-Institute, Postfach
12 01 61, Columbusstrasse, D-27568 Bremerhaven, Germany
* Author for correspondence at present address: International University of Bremen, School of Engineering and Science, PO Box 750561, D-28725 Bremen, Germany (e-mail: m.zakhartsev{at}iu-bremen.de)
Accepted 8 September 2003
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Summary |
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In conclusion, the strategies of LDH adjustment to seasonal temperature
variations in cod involve changes in LDH concentration (quantitative),
adjustment of thermodynamic (Ea) and kinetic
() properties of the LDH
(modulative) but not the expression of alternative isoforms (qualitative). We
assume that the observed increase in Ea and the decrease
of temperature dependence of
at low
TA is the result of structural changes of the LDH molecule
(temperature-driven protein folding). We propose a new mechanism of metabolic
compensation of seasonal temperature variations cold acclimation
results in changes in the kinetic and thermodynamic properties of LDH in a way
that favours aerobic metabolism through reduction of the competition of LDH
for pyruvate in normoxic conditions.
Key words: allozyme, Arrhenius activation energy, fish, Gadus morhua, glycolysis, isozyme, kinetics, lactate dehydrogenase, LDH, liver, metabolic compensation, muscle, temperature acclimation
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Introduction |
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During adaptation or acclimation to low temperatures, the capacity of
anaerobic metabolism may be subject to adaptive modification. While
compensation of aerobic enzymes and mitochondrial densities is a
well-established feature of cold adaptation (cf.
Pörtner, 2002b for a
review) the picture is less clear for anaerobic capacity. In polar fish such
as Antarctic notothenioids, excess of aerobic design at low activity
lifestyles would explain why anaerobic capacity is generally reduced.
Oxidative enzymes show relatively high degrees of cold compensation in
notothenioids, while glycolytic enzymes do not
(Crockett and Sidell, 1990
;
Dunn and Johnston, 1986
;
Johnston, 1987
). In contrast
to the low lactate levels in fatigued notothenioids, a significant anaerobic
capacity was recently found in Antarctic eelpout (Pachycara
brachycephalum; Hardewig et al.,
1998
), a benthic and rather immobile species. Cold-compensated
anaerobic capacity was also found in some, but not all, temperate freshwater
fish (Van Dijk et al., 1998
).
As a corollary, glycolytic capacity may still be cold compensated in strictly
benthic zoarcids, but no longer in more active polar fishes, which, therefore,
tend to express a more aerobic mode of metabolism. The functional modification
of LDH depending on the ambient temperature regime has been a focal issue in
the study of thermal adaptation, including analyses of substrate affinities,
thermodynamic properties and changes in enzyme structure that may cause the
functional modifications observed
(Danilenko et al., 1998
;
Fields et al., 2001
,
2002
;
Fields and Somero, 1998
;
Hochachka and Somero, 1984
;
Klyachko et al., 1995b
;
Ozernyuk et al., 1994
;
Persikov et al., 1999
;
Sharpe et al., 2001
;
Zasedateleva et al.,
1999
).
The question arises as to whether temperature-dependent changes in
anaerobic capacity or functional properties of LDH can also be seen in
temperate or sub-Arctic to Arctic cold water fish like the cod Gadus
morhua. This demersal species displays moderate levels of aerobic
capacity and uses significant anaerobic metabolism at high, beyond critical,
swimming speeds (Reidy et al.,
1995). Therefore, the present study was designed to investigate
the changes in the capacity and functional properties of LDH in cold-
vs warm-acclimated cod from a temperate population.
In many fishes, LDH is coded by three independent loci
(De Panepucci et al., 1984;
Ferreria et al., 1991
;
Zietara et al., 1996
).
Simultaneous expression of LDH isozymes (A, B and C) and allozymes of LDH-B
yields a complicated electrophoretic pattern (overall LDH suite), which is
sometimes difficult to interpret. The protein (LDH) is a tetramer, and the two
alleles result in a five-band electrophoretic pattern of allozymes (in the
case of a heterozygote; Hillis et al.,
1996
; Utter et al.,
1987
). LDH in cod (Gadus morhua) is represented by three
loci; Ldh-A and Ldh-B have been identified in liver, muscle,
heart and eye, while Ldh-C is only observed in liver
(Mork et al., 1985
;
Zietara and Skorkowski, 1993
).
Although an alternative allele is found in Ldh-A in cod from Iceland
(Mork et al., 1985
),
Ldh-B is the most variable locus for this species. Polymorphism in
locus Ldh-B can be described by two alleles (a and
b, with a relative migration distance of 70 and 100 during gel
electrophoresis with histidine buffer, pH 7.0) in cod and is mainly
represented by three common phenotypes; Ldh-B(a/a),
Ldh-B(a/b) and Ldh-B(b/b). In Norwegian
waters, however, two rare alleles Ldh-B(c) (with a relative
migration distance of 85) and Ldh-B(d) (with a relative
migration distance of 140) have been observed as rare heterozygotes, combined
with the allele Ldh-B(b)
(Mork et al., 1985
).
Some polymorphic loci display a latitudinal cline in allele frequency,
which correlates with a change in mean water temperature
(Hummel et al., 1997;
Kitto et al., 1983
;
Powers and Place, 1978
;
Powers and Schulte, 1998
).
This observation indicates the adaptive significance of changes in allozyme
composition for ectothermic animals under particular environmental conditions.
Highly significant heterogeneity between areas was found at the Ldh-B
locus, which distinguished North-Eastern Arctic cod (NEAC) from other cod
stocks (Mork et al., 1985
).
Ldh-B may also differ between Norwegian coastal cod and NEAC
(Jorstad, 1984
;
Mork and Giaever, 1999
)
together with differences in other genetic markers like haemoglobin,
phosphoglucose isomerase (Dahle and
Jorstad, 1993
; Jorstad,
1984
; Mork et al.,
1985
) and synapophysin
(Fevolden and Pogson,
1997
).
Although it is more common for isozymes to show distinct differences in
functional characteristics than for enzymes coded by alternative alleles
(allozymes), the latter may also differ in their kinetic properties
(Henry and Ferguson, 1987;
Jollivet et al., 1995
;
Place and Powers, 1984
). In
some circumstances, these kinetic differences could lead to
frequency-dependent selection, which is potentially capable of maintaining
balanced polymorphism (Clarke and
Allendorf, 1979
). In the present paper, we looked at LDH
acclimation capacity in cod, including aspects of acclimation from the
population level down to the thermodynamic level of the enzyme. We have chosen
to work on crude homogenates (e.g. with the overall LDH suite), because it is
well known that purified LDH-B allozymes have significant kinetic differences,
as shown in the mummichog Fundulus heteroclitus
(Place and Powers, 1984
).
Accordingly, the following questions were addressed: what are the implications
of changes in LDH-B allozyme content for the final functional properties of
the overall LDH suite in cod, and which mechanisms are involved in modifying
LDH activity during seasonal temperature variation?
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Materials and methods |
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Acclimation
The cod were tagged individually with pit-tags and then randomly separated
into three groups and acclimated to 4°C (two tanks) and 12°C (one
tank) in a flow-through system with natural seawater. The water for the
experiment was pumped from 100 m depth of the fjord outside Bergen, where
salinity and temperature remain constant throughout the year. The 4°C
acclimation groups consisted of 39 and 41 fishes in each tank (80 in total),
and the 12°C acclimation group comprised 42 animals. 4°C is a common
temperature for cod in boreal regions, and 12°C is close to the optimal
temperature for somatic growth of cod
(Jobling, 1988
;
Pörtner et al., 2001
).
Temperature was maintained at ±1°C during the experimental period
from 19 January 1999 until 7 March 2000 (408 days). The fishes were fed in
excess with commercial dry pellets for marine fishes (NorAqua, Trondheim,
Norway) by an automatic feeder. A simulated natural light regime was used
corresponding to ambient light conditions in Bergen (60°N). The oxygen
concentration in the outlet water was recorded daily and remained above 86%
saturation. Water salinity was 33
throughout the experiment.
Physiological conditions
The growth rate was measured individually once every six weeks, and
condition factor (k-factor), hepatosomatic index (HI), and
gonadosomatic index (GI) were measured at the end of the experiment. The rate
of oxygen consumption
(O2; µmol
min1 g1) was measured in a flow-through
respirometer using the microoptode technique
(Van Dijk et al., 1999
).
O2 data that are
presented in the article have been collected in a separate acclimation
experiment on Southern North Sea cod in the Alfred Wegener Institute
Foundation for Polar and Marine Research (AWI; Bremerhaven, Germany) by T.
Fischer (unpublished data; N=8).
Sampling
At the end of the experiment (7 March 2000) the fishes were killed by the
addition of MS222 (tricaine methane sulphonate) to the water (1 g
l1). Total fresh mass, length, liver mass and gonad mass
were measured, and tissues (blood, white muscle and liver) were sampled. The
sampling time was minimised and it generally took less than 3 min from the
killing of the fish until sample fixation. The samples were freeze clamped in
liquid nitrogen (196°C), transported on dry ice (78.5°C)
and stored in a deep freeze at 80°C.
Genetic variation
According to previous studies on genetic variation in Atlantic cod, the
allozyme of hemoglobin (Hb-I) from blood and five allozyme markers (LDH-B,
GPI, GPD, IDH and PGM) from white muscle were chosen as markers for population
genetic analysis (Gjosaeter et al.,
1992; Jorstad,
1984
; Mork and Giaever,
1999
; Renaud et al.,
1986
). Genetic variation in Hb-I was freshly analysed on agar gel
in Smithies buffer pH 8.6, as described by Fyhn et al.
(1994
). The muscle allozyme
markers were analysed by starch gel electrophoresis, performed in histidine
buffer pH 7.0 and stained as described by Jorstad
(1984
). The allozyme
frequencies were analysed by computer program BIOSYS-1 (University of
Illinois).
LDH isoelectric focusing
Both starch and agar gel electrophoresis were used to detect allele
frequencies in six selected loci (including Ldh-B). However, later
the samples were screened again by isoelectric focusing (IEF) but only for
LDH. The results of allozyme identification were completely identical between
methods.
20% tissue homogenates (w/v) were prepared (Ultra-Turrax T25) in ice-cold
20% sucrose water solution, and then the samples were centrifuged at
15x103 g for 20 min. Supernatant was
collected and used for IEF. IEF was carried out in PhastSystem (Amersham
Pharmacia Biotech, Uppsala, Sweden) at 6°C. The samples (1 µl) were
applied in the middle of PhastGel IEF 3.59.5 (Amersham Pharmacia
Biotech). The focusing was carried out using the standard pH 39
protocol provided by Amersham Pharmacia Biotech: pre-focusing (75 AVh at 780
V, 1.5 W, 2.2 mA, 6°C); focus with applicator (100 AVh at 200 V, 0.1 W,
0.4 mA, 6°C); focus after removal of the applicator (480 AVh at 2000 V,
3.5 W, 2.5 mA, 6°C). LDH staining solution for one PhastGel contained 10
ml of 131 mmol l1 D/L-lactic acid
(racemic liquid mixture) in 0.2 mol l1 Tris pH 8.0; 250
µl NAD+ (10 mg ml1); 250 µl MTT
(methyl-thiazolyl blue; 5 mg ml1); 125 µl PMS (phenazine
methosulphate; 5 mg ml1)
(Hillis et al., 1996).
Staining solution was freshly prepared and all staining procedures were
performed in the dark at room temperature. Staining time was between 10 min
and 15 min depending on the appearance of the bands but before the background
got too dark.
We used an IEF calibration pI kit (pH 3.59.3; Amersham Pharmacia Biotech). The calibration markers were always applied to the first and the last lanes of each gel. After focusing they were cut off and stained separately according to the `fast Coomassie staining protocol for PhastGel IEF' provided by Amersham Pharmacia Biotech (development technique file No 200; PhastSystem). After the gel was dried it was scanned (with a regular scanner), and the isoelectric points (pIs) of every single band on the gel were calculated using computer program ImageMaster TotalLab V1.11 (Amersham Pharmacia Biotech).
Ldh locus classification (Ldh-A, -B and -C) was
adopted from Grant and Stahl
(1988) and Zietara and
Skorkowski (1993
).
LDH kinetic studies with saturating cofactor levels
LDH is an equilibrium enzyme, which catalyses the reaction:
![]() | (1) |
1% tissue homogenates (w/v) were prepared (Ultra-Turrax T25) in ice-cold
buffer (Hepes, 100 mmol l1 KCl, pH 7.00 at 20°C) and
then the homogenates were centrifuged at 15x103
g for 20 min at 4°C
(Yang and Somero, 1993). The
supernatant was used for kinetic analyses. The concentration of the enzyme was
adjusted to be sufficiently low that the reaction proceeded at a constant rate
over kinetic time and caused a decrease in optical density by less than 10% of
initial values. The kinetics were read (every 15 s for 3 min) using Bio-Rad
(Hercules, CA, USA) 3550-UV microplate reader set at 340 nm. The sample was
shaken for 2 s before each reading. In order to keep assay conditions within
the limits of linearity at the different temperatures, the dilution factor of
the 1% homogenate was increased correspondingly (for muscle, from 50 at
4°C to 500 at 30°C; for liver, from 11 at 4°C to 100 at 30°C).
To collect statistically sufficient data on the temperature dependence of the
enzyme kinetic parameters, we designed a temperature-controlled 96-well
microplate for the Bio-Rad 3550-UV microplate reader
(Zakhartsev and Blust, 2002
).
This system allows for a highly accurate (±0.1°C) on-line control
of temperature in the microplate over a broad temperature range (from 0°C
to 60°C).
The reaction was started by mixing 100 µl of diluted homogenate with 200
µl of stock solution of the assay medium pre-set to the desired
temperature. The reaction was started directly in the 96-well
temperature-controlled microplate placed in the microplate reader. A 30 s time
delay with constant shaking was used prior to the beginning of the reading to
allow the reaction solution to mix and reach the assay temperature; only then
was the reaction monitored for 3 min. The final volume of the assay was 300
µl containing 200 µmol l1 ß-NADH (this is the
saturated level for LDH of marine fish;
Place and Powers, 1984),
variable pyruvate levels (05 mmol l1) and 100 mmol
l1 KCl in 50 mmol l1 Hepes, pH 7.0 (at
20°C). Enzyme activity was always determined in triplicate at 10 pyruvate
concentrations (0, 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3.5 and 5 mmol
l1; see Fig.
1) and at seven assay temperatures (4, 8, 12, 16.5, 21, 25.5 and
30°C; see Fig. 2A). Thus,
30 enzyme assays were performed on each individual fish at one assay
temperature, yielding 210 different assays for seven different assay
temperatures.
|
|
It is well known that LDH activity is inhibited by high substrate
concentrations (substrate inhibition kinetics;
Fig. 1;
Table 1); therefore, at
saturating levels of the cofactor (NADH) the reaction rate is described by:
![]() | (2) |
|
Nonlinear regression analysis (GraphPad Prism software) was used to
calculate all kinetic parameters and fit the curve. The actual maximal
reaction rate (max) always falls short of
Vmax (from equation
2; open circle in Fig.
1). Therefore, the substrate concentration at which the reaction
rate is maximal can be calculated by differentiating
with respect to
[S] and finding a value of [S] at which the slope of the
curve is zero ([S]max)
(Cornish-Bowden, 1999
):
![]() | (3) |
![]() | (4) |
The specific LDH activity was expressed as µmol of NADH oxidized per
minute per mg of total protein (U mg1 protein) in the
reaction mixture using 6.22x103 O.D. mol1
cm1 as the extinction coefficient of NADH
(Dawson et al., 1986).
NanoOrange® Protein Quantitation Kit (Molecular Probes, Eugene, OR, USA)
was used to determine the total protein concentration in diluted
homogenates.
LDH thermodynamic studies
The calculation of the apparent Arrhenius activation energy
(Ea) for enzymatic reactions is commonly based on rate
measurements made at saturating substrate concentrations (i.e.
Vmax), when substrate availability will not be limiting
for the reaction rate (Segel,
1976). However, in cases of high-substrate-inhibition kinetics,
the calculated Vmax never coincides with
max (Fig. 1; Table 1). Thus, we used
temperature dependence of
max (from
equation 4; Fig. 2A open circles) to
calculate Ea, since it is closer to the real rate values
at saturated conditions. The Ea (J mol1)
was calculated from the slope of the Arrhenius plots
[Ea=slopexR, where R is the
universal gas constant (J mol1 K1)].
The change in activation enthalpy (H; J
mol1) was calculated as follows:
![]() | (5) |
The change in activation entropy (S; J
mol1 K1) was derived from
`enthalpyentropy compensation plots'
(Hochachka and Somero, 1984
;
Prosser, 1986
). The
compensation plot (Fig. 2B) was
compiled from published data for purified LDH according to the concept
outlined in Hochachka et al.
(1976
), Prosser
(1986
) and Cornish-Bowden
(2002
). Gibbs free energy
change (
G) was computed according to the following
(Prosser, 1986
):
![]() | (6) |
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Results |
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Growth performance
Two-way analysis of variance (ANOVA) revealed a significant effect of
acclimation temperature on growth performance in terms of both length and mass
(P<0.001) but did not reveal an effect of Ldh-B
phenotypes on growth performance (Table
3). Interestingly, during the summer period, growth rate was
markedly different between acclimation groups. However, after November 1999,
daily growth rate became similar at the two acclimation temperatures
(Fig. 3). This is a common
phenomenon for maturing cod. A significant decrease in growth rate in November
might be caused by physiological changes in preparation for spawning in the
spring of the following year. These patterns indicate that not only
acclimation temperature but also probably daylight duration (season) and
maturation stage have an effect on growth rate (multiple-factor effect);
however, Ldh-B phenotypes give no advantage for the growth
performance.
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|
Further analysis did not reveal any effect of Ldh-B phenotypes nor
acclimation temperature on k-factor and hepatosomatic index (HI),
indicating that the masslength relationship remained unaffected by the
acclimation temperature. However, the gonadosomatic index (GI) was
considerably higher (4.25) after acclimation to 4°C compared with 12°C
acclimation (2.68) (Table 3). Rates of oxygen consumption
(O2) of
cold-acclimated (4°C) cod from the North Sea were significantly higher
(t-test, P<0.05) than those of warm-acclimated cod
(12°C), even when the comparison was carried out at their acclimation
temperatures (Table 3).
However, the Arrhenius activation energy for
O2 was lower in
4°C-acclimated cod (33.9 kJ mol1) than in
12°C-acclimated cod (38.4 kJ mol1) (T. Fischer et al.,
unpublished data on Southern North Sea cod).
Isoelectric focusing
Only the Ldh-B locus displayed a protein polymorphism due to two
alleles, Ldh-B(a) and Ldh-B(b), that give three
corresponding phenotypes (Fig.
4). The tetrameric LDH gives the classical five-band pattern (with
pI values between 5.16 and 5.45; Table
4; Fig. 4) for the
Ldh-B heterozygote [Ldh-B(a/b)]. As Ldh-B was the
only polymorphic locus among Ldh loci in cod, and since it was
equally expressed in both tissues, we used this distinctive locus to identify
LDH electrophoretic patterns (overall LDH suites). We worked on crude
homogenates where several Ldh loci and alleles were simultaneously
present. Therefore, the particular Ldh-B phenotype actually includes
the overall LDH pattern (Fig.
4).
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|
Detailed analysis of isoelectric focusing patterns revealed distinctive
differences between the two tissues (Fig.
4). Ldh-B and Ldh-A loci were expressed in both
tissues, while the Ldh-C locus was only expressed in liver
(Fig. 4). The Ldh-A
locus in muscle showed a complicated pattern of bands with pIs between 6.0 and
6.3. At the same time, LDH-A in liver displayed only one band with a pI
equivalent to the pI of the central LDH-A band in muscle (pI 6.15;
Table 4). These observations
indicate that the sub-bands in muscle LDH-A represent an artefact rather than
allozyme variability. In order to see more clearly the minor LDH-B bands we
were forced to increase the protein concentration in applied muscle samples,
which resulted in an overloading of the major LDH-A band. This may have caused
the development of sub-band artefacts. It is a common observation that samples
from different tissues with similar enzyme expression patterns, when
overloaded, exhibit differences in the formation of sub-bands during
electrophoresis (Richardson et al.,
1986).
The bands with pI values between 5.65 and 5.86 that were located between LDH-A and LDH-B are probably interlocus hybrid bands. Therefore, they appeared in different positions in homozygotes [for Ldh-B(a/a) pI=5.85; for Ldh-B(b/b) pI=5.65] and heterozygotes [Ldh-B(a/b) pI=5.655.85].
Locus Ldh-C (pI 8.3) and intermediate bands (pI 7.4 and pI 7.7)
appear to be unique for the liver of cod, as shown previously
(Mork et al., 1985). A
monomorphic band at pI 7.15 (band No 4 in
Table 4) is present in both
tissues; however, this isozyme has never been described before, although it
appears as a major LDH isozyme in liver. Quantitative analysis of the
composition of the LDH suite showed that products of the polymorphic locus
(Ldh-B) represent only
14% of the total LDH suite in muscle and
19% in liver.
Image analysis and statistical analysis of the enzyme patterns (ANOVA) obtained by IEF (Table 4) revealed distinct differences between tissues in LDH isoelectric patterns but did not reveal any effect of acclimation temperature on the appearance of new LDH isoforms and on the pI of the corresponding LDH bands (P>0.05). In other words, we found no evidence that acclimation to different temperatures has any effect on the composition of the LDH suite and the quaternary structure of the LDH enzymes.
LDH kinetic studies
All kinetic studies of the overall LDH suite in crude homogenates were
performed at saturating levels of the cofactor (NADH). The kinetic studies of
LDH in crude homogenates demonstrated the well-known substrate inhibition of
LDH by pyruvate (Fig. 1; Table 1). We could not identify
differences between Ldh-B phenotypes (overall LDH suites) in any of
the kinetic parameters (Vmax,
and
), which were measured at
both acclimation temperatures (ANOVA, P>0.05;
Table 5). However, there was a
distinct difference between tissues (Fig.
1; Table 1). Liver
LDH in crude homogenate displayed a lower
and
than did muscle LDH and,
consequently, higher relative catalytic efficiencies of the reaction at
physiological substrate concentrations
(
/
;
Fig. 1;
Table 1).
|
The presence of Ldh-B phenotypes did not cause significant
differences between levels and temperature dependencies of
Vmax,
and
of LDH in crude
homogenates (F-test, P>0.05). Thus, in terms of reaction
kinetics, we could not find significant differences between overall LDH suites
in crude homogenates that might be associated with different Ldh-B
phenotypes. Therefore, the kinetic data from different phenotypes were pooled
for further analysis and presentation.
The analyses of the temperature dependence of kinetic parameters were performed for values between 4°C and 30°C. An assay temperature of 30°C already results in a significant deviation of activities from the general trend. This conclusion originated from the observation that the values of r2 for exponential curves became considerably higher once the rate values at 30°C were omitted. At the same time, the absolute sum of square (ASS) and the standard deviation of the residuals (Sy.x) became considerably lower. Such omission increased r2 to up to 0.99. Additionally, the high values of r2 indicate that the chosen exponential function adequately fits the data. Consequently, the rate values at 30°C were omitted from further calculations of temperature dependence of kinetic parameters and thermodynamic parameters (Figs 5, 6, 7).
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max and
Comparison of the temperature dependencies of max per mg
of total tissue protein (U mg1 protein;
Fig. 5A) showed that muscle
displayed significantly higher specific LDH activities in comparison with
liver in the 12°C acclimation group (F-test, P<0.01).
Unpaired t-tests revealed that a quantitative component of the
exponential equation became significantly higher in the 12°C acclimation
group. As
max is a relative measure of enzyme concentration
{
max is in direct proportion to Vmax
(equation 4), but
Vmax=kcat[E]t},
then we can conclude that the specific concentration of LDH in muscle was
reduced in response to cold (4°C), probably as the result of an increased
aerobic capacity (see Introduction). However, recalculation of
max in terms of activities per g wet mass (U
g1 wet mass; Fig.
5B) showed almost complete compensation of the final enzyme
activity.
Analysis of at
acclimation temperature (ANOVA, P>0.05) and its temperature
dependence (F-test, P>0.05) did not reveal a significant
difference among Ldh-B phenotypes, which is why the data were pooled.
Further analysis did not reveal significant differences between
values for LDH in crude
homogenate at the two acclimation temperatures (F-test for entire
curves, P>0.05; Fig.
6). The change in
with temperature showed
an exponential pattern resulting in a lower thermal sensitivity at lower
temperature. Two-phase linear regression analysis showed that at temperatures
below 16±1°C,
displays low temperature dependence, with a slope of 0.0030.008 mmol
l1 deg. (Fig.
6).
Analysis of at
corresponding acclimation temperature (ANOVA, P>0.05) and its
temperature dependence (F-test, P>0.05) did not reveal a
significant difference between Ldh-B phenotypes. At corresponding
acclimation temperatures,
was higher in white
muscle than in liver (Table 5).
of both tissues
underwent a significant change over the course of acclimation to different
temperature (F-test, P<0.05;
Fig. 7). Acclimation to low
temperature resulted in a lowering of
values compared with
acclimation to high temperature, as well as a decrease in the
temperature-dependent increment of
in both tissues.
Analysis of
/
(when Vmax was expressed in U mg1 total
protein) at corresponding acclimation temperatures did not reveal a
significant difference among Ldh-B phenotypes (ANOVA,
P>0.05; Table 5).
Nevertheless, the efficiency of the LDH suite is higher in liver than in
muscle, when expressed in terms of Vmax per mg protein
(Table 5). However, when
/
was expressed in terms of total Vmax per g of wet tissue
(U g1 wet mass), the final LDH efficiency became similar in
both tissues at corresponding acclimation temperatures
(Table 5;
Fig. 8). This means that,
despite a considerable difference between the total mass of muscle and liver,
the total LDH efficiency in each tissue is similar. Moreover, acclimation to
different temperatures results in partial compensation of total
/
in muscle and almost complete compensation in liver (unpaired t-test;
Fig. 8).
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Total protein concentration
The concentration of total tissue protein was not different among
Ldh-B phenotypes (ANOVA, P>0.05). However, it was
increased during acclimation to low temperature
(Table 5) from 36.6 mg
g1 wet mass to 47.0 mg g1 wet mass in
muscle and from 23.4 mg g1 wet mass to 29.7 mg
g1 wet mass in liver. In terms of max (U
mg1 protein), the LDH-specific concentration was reduced
under acclimation to low temperature, but in terms of final
max per tissue (U g1 wet mass) the total
LDH-specific concentration was restored by quantitative compensation after
acclimation to low temperature.
LDH thermodynamic studies
The temperature dependence of max between 4°C and
25°C was used for the calculation of Ea, since values
of
max at 30°C already indicate partial inactivation of
the LDH activity. There were no significant differences in
Ea values for LDH suites between Ldh-B phenotypes
(two-way ANOVA, P>0.05; Table
6). Ea values were very similar in both
tissues (
47 kJ mol1) at 12°C acclimation, despite
very different isozyme patterns and significant differences in kinetic
properties between tissues (Figs
1,
4;
Table 1). This means that
pyruvate reduction in both tissues has the same thermodynamic requirements
despite very distinct differences between tissues both in isozyme patterns and
kinetic properties. However, at 4°C acclimation, the
Ea significantly increased (to
53 kJ
mol1 in liver and to
57 kJ mol1 in
muscle; two-way ANOVA, P<0.05;
Table 6).
|
Analysis of activation enthalpies (Table
6) did not reveal any effect of Ldh-B phenotypes. We have
observed a difference ( in Table
6) in
H between acclimation groups by
5.638.99 kJ mol1 (depending on tissue). This
difference is much higher than would be expected from
equation 5, indicating that the
observed change in
H (
) has another cause than just
temperature dependence of
H under the condition of constant
Ea.
Statistical analysis of Gibbs free energy change values (G)
did not reveal any effect of acclimation temperature, tissue or Ldh-B
phenotype on the level of
G, which remained more or less
constant at
55.5 kJ mol1
(Table 6).
![]() |
Discussion |
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The classification of Ldh loci (A, B and C) was
taken from Grant and Stahl
(1988) and Zietara and
Skorkowski (1993
), while the
Ldh-B phenotype classification followed the general practice in
population genetics to name multiple alleles of a specific locus by lower-case
letters with parentheses (Hillis et al.,
1996
). The Ldh-B locus showed the presence of two
alleles, Ldh-B(a) and Ldh-B(b), yielding three phenotypes:
Ldh-B(a/a), Ldh-B(a/b) and Ldh-B(b/b). We did not use a
classification such as LDH-Ba4,
LDH-Ba/Bb and LDH-Bb4, because we
refer to the overall LDH pattern under Ldh-B phenotypes (i.e. the
mixture of all isozymes and allozymes in crude homogenates the overall
LDH suite), while the latter classification only refers to the subunit
composition of purified allozymes (Place
and Powers, 1984
).
Initially, starch or agar electrophoresis was the main analytical tool for
analysis of population genetics. This method resulted in a number-based
classification that relied on the sequential appearance of isozymes on a gel
(1, 2, 3, etc.) and, thus, the relative mobility of a protein on the gel
(Shaklee et al., 1990). For
the sake of comparability between current and previous classifications, we
have to note that locus Ldh-A corresponds to Ldh-2, Ldh-B to
Ldh-3, and Ldh-C to Ldh-1. In the same way, the
Ldh-B allele a corresponds to 70, and b to 100
units of relative mobility.
In the present study, we found that the loci Ldh-A and
Ldh-B were expressed in skeletal muscle, while all three Ldh
loci were found in liver. Grant and Stahl
(1988) investigated both
Atlantic and Pacific cod isozymes and observed LDH-B in eye fluids and cardiac
muscle as well. The products of the polymorphic locus Ldh-B
contribute only 14% to the total LDH in skeletal muscle and 19% in liver.
Correspondingly, the products of the remaining Ldh loci comprised
86% of the total LDH quantity in muscle (mostly Ldh-A) and
81% of total LDH in liver (both Ldh-A and Ldh-C with
some non-classified proteins).
Zietara and Skorkowski
(1993) analysed cod tissue by
IEF and found that in Gadus morhua from the Baltic Sea the products
of Ldh-A and Ldh-B loci are predominant in both skeletal and
heart muscle (bands with pI=6.4 and pIs from 5.1 to 5.6, respectively), while
products of the Ldh-C locus predominate in liver (band with pI=8.2).
The bands with pIs of 7.64, 7.10 and 6.66 seen in our measurements have not
been reported before, however; these bands represent a large fraction of the
liver's overall LDH suite (11%, 26% and 11% of total LDH, respectively; 48% in
total). This pattern raises a problem for the precise classification of LDH
proteins in liver of our cod, as some proteins have not been previously
described. In order to avoid these complications, we preferred to operate with
the overall LDH pattern (the LDH suite), qualified according to the
polymorphism in locus Ldh-B.
Analysis of the IEF patterns did not reveal differences in the number of
bands between different acclimation groups. This indicates that acclimation
temperature did not cause the expression of new LDH isozymes. Earlier,
Ozernyuk et al. (1994) and
Vornanen (1994
) reported the
same phenomenon for LDH of loach (Misgurnus fossilis) and crucian
carp (Carassius carassius) during seasonal temperature variations.
Thus, Atlantic cod does not exploit the so-called qualitative adaptation
strategy, i.e. the expression of new LDH isozymes that suit a new
environmental condition better.
Quantitative analysis of pIs did not reveal an effect of acclimation
temperature on enzyme mobility. This means that the quaternary structure of
LDH was not affected by acclimation. Previous electrophoresis studies
(Ozernyuk et al., 1994; G.
Nævdal, personal communication) did not reveal any difference in LDH
mobility between individuals acclimated to different temperatures. Thus, based
on the unchanged frequencies of the Ldh-B allele after acclimation
and on unchanged isozyme patterns and enzyme mobilities, we can conclude that
cod pass through the seasonal temperature cycle with genetically the same LDH
suite.
The cod that displayed different Ldh-B phenotypes did not reveal
differences in growth performance or physiological condition. This is in
accordance with findings by Nævdal et al.
(1992), who carried out a
similar growth experiment for cod at three environmental temperatures
(6°C, 10°C and 12°C), where they showed that the growth rate was
significantly affected only by acclimation temperature. Acclimation
temperature had no effect on HI and k-factor, indicating that the
fish just grew slower at cold temperature without any resulting anomalies
(deviation from masslength relationship). At first sight, this may
simply appear as a consequence of reduced metabolic rate in the cold; however,
we found that the temperature-specific rate of oxygen consumption
(
O2) was higher
in cold- than in warm-acclimated fish, which appears typical for temperate
eurythermal marine fish (Van Dijk et al.,
1999
; Zakhartsev et al.,
2003
). The observations that growth performance was reduced and
aerobic metabolic rates increased with decreasing acclimation temperature
indicate metabolic reorganization during cold acclimation. Under laboratory
conditions, the decrease of growth rate in the cold may be a consequence of
high baseline costs of cold adaptation, in line with a hypothesis developed by
Pörtner et al. (2001
). An
enhanced level of aerobic metabolism may be paralleled by a decreased capacity
of anaerobic metabolism and modified functional properties of LDH. In the
following, LDH functional properties will be discussed in this context.
Kinetic study
Hepes was used as a medium buffer because it displays a
temperature-dependent pH slope of 0.015 pH deg.1
(Dawson et al., 1986;
Yancey and Somero, 1978
),
which is very close to that of the pattern of intracellular pH change in some
marine fish (e.g. common eelpout Zoarces viviparus;
Van Dijk et al., 1999
). At the
same time, in contrast to imidazole, Hepes does not interfere with the protein
quantification method we used (NanoOrange®).
Despite the extensive use of LDH in analyses of population genetics and
biochemical adaptation, only a few publications deal with comprehensive
kinetic studies of LDH (including iso- and allozymes) from ectothermic
animals; for example, see Place and Powers
(1984) and Sharpe et al.
(2001
). It is well documented
that LDH from most ectothermic animals displays clear substrate inhibition
kinetics (Almedia-Val et al.,
1991
; Baldwin et al.,
1989
; French and Hochachka,
1978
; Narita and Horiuchi,
1979
; Place and Powers,
1984
; Tsukuda and Yamawaki,
1980
; Zietara et al.,
1996
; Zietara and Skorkowski,
1993
), caused by the formation of a covalent adduct between
pyruvate and the oxidized form of the cofactor, i.e. the formation of an
inhibitory enzymeNAD+pyruvate complex
(Eszes et al., 1996
;
Hewitt et al., 1999
). LDH in
crude homogenates of cod tissues also showed substrate inhibition. LDH is an
equilibrium enzyme, but in our research we studied only the rate of pyruvate
reduction, since pyruvate is the key substrate for both aerobic and anaerobic
branches of the metabolism.
It has been shown several times that purified LDH allozymes differ
significantly in their kinetic properties
(Henry and Ferguson, 1986;
Hoffmann, 1981
;
Place and Powers, 1984
;
Zera, 1987
;
Zietara and Skorkowski, 1993
).
However, since all LDH isoforms operate simultaneously in the cytoplasm
(Voet and Voet, 1995
), each
isoform of LDH should play a particular role in the overall performance of
LDH. This is the reason why we used crude homogenates for our kinetic studies.
Nevertheless, the question arises of what effect the LDH-B allozymes have on
the overall performance of the LDH suite in the cytoplasm. Thorough analysis
of the temperature dependence of kinetic parameters (Vmax,
and
) did not reveal any
difference between Ldh-B phenotypes (overall LDH suites). As LDH-B
comprises only 1419% of all LDHs present in the crude homogenate,
differences between these allozymes have no significant consequences for the
final kinetic parameters of the LDH suite in the cytoplasm and, hence, for the
net performance of the LDH suite.
We observed very significant tissue-specific differences in the kinetic
properties of LDH suites related to different isozyme patterns. Since the
different pI patterns in the two tissues were completely due to the presence
of isozyme Ldh-C and to unclassified isozymes with pIs of 7.64, 7.10
and 6.66, the tissue-specific kinetic difference must be caused by the
presence of these isozymes but not by the allozymes of LDH-B. According to
Zietara and Skorkowski (1993),
LDH-B allozymes are among those LDH isozymes most sensitive to high substrate
concentrations. Consequently, LDH-B allozymes could serve as sensitive control
elements in complex multi-isozyme LDH ensembles.
Comparison of tissue-specific kinetic parameters at different acclimation
temperatures showed that muscle and liver LDH suites have distinctively
different kinetic profiles (Fig.
1;Table 1). Muscle
LDH displayed higher values of all kinetic parameters
(Vmax,
and
), which indicates that
this LDH suite is designed to work at higher, and over a wider range of,
pyruvate concentrations than liver LDH. This allows a higher substrate flux
via LDH and thereby supports a higher rate of anaerobic glycolysis
and, at the same time, fine control at elevated flux rates (higher pyruvate
levels). The higher relative catalytic efficiency
(
/
)
in liver indicates that this LDH suite is designed to work faster and more
efficiently under conditions of lower substrate concentrations. Possibly,
these are lowered by higher tissue aerobic capacities when pyruvate is readily
oxidized by mitochondria. Since LDH is an equilibrium enzyme, we can also
conclude that the observed tissue peculiarities reflect different biochemical
roles of LDH in the two tissues. On the one side, anaerobic muscular work
relies on the glycolytic pathway under conditions of higher metabolite flux as
well as elevated lactate levels. On the other side, liver is a sink for
lactate, and consequently LDH operates efficiently at lower lactate and
pyruvate levels in this organ (Cori cycle;
Voet and Voet, 1995
).
Neither acclimation temperature nor Ldh-B phenotype had an effect
on for the overall LDH
suite. Since the affinity to the substrate
(
) is a major
`fingerprint' of the enzyme, we can conclude that the functional properties of
the LDH suite remained the same after acclimation to different temperatures.
Although the temperature dependence of
followed an exponential
curve (Fig. 6), we decided to
apply a two-phase linear regression analysis to this data set, because it is
obvious that
displayed a
very weak temperature dependence in the low temperature range
(Fig. 6). This analysis
revealed that at temperatures below 16±1°C (which was found to be
the breakpoint)
displayed
a very low temperature dependence with a slope of 0.0030.008 mmol
l1 deg.1. It is remarkable that both
tissues at both acclimation temperatures showed the same breakpoint at
16°C. This has important implications for the capacity of anaerobic
metabolism. The loss of substrate affinity (increase of
beyond this breakpoint)
results in a change of the enzyme's `reserve capacity' to control metabolic
flux (Hochachka and Somero,
1984
).
According to Fig. 5, the specific concentration of LDH was reduced in both tissues during acclimation to cold temperatures. By contrast, the rise in total protein concentration (Table 5) reflects an increase in the concentration of functional proteins to compensate for the loss in functional capacity of the tissues in the cold (Fig. 5).
The ratio
/
reflects the relative catalytic efficiency of an enzyme, which represents a
rate constant at physiological substrate concentrations; therefore, to some
extent, it is a measure of enzyme efficiency in vivo
(Place and Powers, 1984
).
Acclimation to low temperature resulted in a significant decrease of enzyme
efficiency in both tissues [
/
;
Table 5]. However, due to the
increase in overall tissue protein levels with acclimation to low temperature,
the total tissue LDH activity [
/
] was
almost completely compensated in liver and partially compensated in muscle
(Fig. 8). Interestingly, the
magnitudes of total tissue LDH efficiencies
(
/
)
are similar in both tissues (Fig.
8). This finding probably reflects biochemical tasks of the
tissues (e.g. in the Cori cycle, which, however, is of little relevance in
fish). There are three main variables that support the accomplishment of such
compensation: LDH-specific concentration, total protein concentration and
total mass of the organ. Therefore, we can conclude that, at both acclimation
temperatures, pyruvate reduction by LDH (when calculated per organ wet mass)
displayed similar catalytic efficiencies at physiological substrate
concentrations in liver, and the compensation in muscle was not completed.
Total LDH efficiency was somewhat reduced in muscle, possibly as a consequence
of the cold-induced metabolic reorganization towards aerobic metabolism
(Lannig et al., 2003
).
Place and Powers (1984)
showed that a high degree of substrate inhibition is typical for both
directions of the reaction catalysed by LDH. In the marine teleost
Fundulus heteroclitus, substrate inhibition at 25°C was observed
at
mmol
l1 and at
mmol
l1. The latter matches very well the level of
found in the present
study in cod.
is another
distinctive feature to characterize the capacity of LDH to cope with increased
metabolic flux. Acclimation to low temperature significantly reduced
in both tissues,
indicating enhanced inhibition, and
also became less
dependent on assay temperature (Fig.
7). Narita and Horiuchi
(1979
) also found that
in muscle LDH of
crayfish (Procambarus clarki) fell during acclimation to low
temperature (from 0.24 mmol l1 at 25°C to 0.09 mmol
l1 at 5°C). The substrate inhibition phenomenon is
caused by the formation and dissociation of a covalent adduct between pyruvate
and the oxidized form of the cofactor. The Ser163 amino acid residue plays a
key role in this mechanism (Eszes et al.,
1996
; Hewitt et al.,
1999
), as well as the presence of several active sites and subunit
interactions within the tetrameric protein. Consequently, the change in
temperature dependence of
reflects changes in the
formation and dissociation of a covalent adduct between pyruvate and the
oxidized form of the cofactor, probably as a result of an altered interaction
between LDH subunits. By contrast,
remained unaffected by
temperature acclimation. The difference between
and
(
=
)
characterizes the range of pyruvate concentrations where LDH can work at
maximal capacity. This range shrank significantly in both tissues with
acclimation to low temperature, emphasising a reduction of anaerobic scope at
low temperature (4°C). This also means that at low acclimation temperature
(4°C) the capacity of LDH becomes more limited
(
=
)
to support rapid turnover of high pyruvate concentrations. This is indicative
of restricted capacity of anaerobic glycolysis after acclimation to low
temperature (Pörtner,
2002b
).
Thermodynamics
To perform the large number of enzymatic analyses in the temperature range
between 4°C and 30°C, which is required for the thermodynamic
characterisation of the enzyme from wild animals, we have designed a
temperature-controlled 96-well microplate to employ a microplate reader for
the purpose of high-throughput screening (see N and n in
Table 6;
Zakhartsev and Blust, 2002).
Usually, studies of the temperature dependence of enzyme kinetic parameters
and thermodynamics from individuals are limited in terms of group sizes
(maximum, 23 animals), mainly because of the technical limitation of
the corresponding equipment (M. Zakhartsev, H. O. Pörtner and R. Blust,
unpublished). Now, this methodological limitation has been overcome and allows
more robust statistical comparisons of enzymatic Ea
between acclimation groups (see N and n in
Table 6).
The classical view on changes in Ea with thermal
adaptation or acclimation is that Ea decreases in the cold
in order to compensate for the effect of decreasing temperature on reaction
rates (Hochachka and Somero,
1984). Our finding that Ea of LDH can increase
with a decrease in acclimation temperature is in contrast to this concept.
However, our observation is unequivocal and supported by a large dataset (see
n in Table 6).
Moreover, a number of more recent examples has shown that a decrease of
Ea in the cold is not necessarily a unifying trend
(Pörtner et al., 2000
).
Van Dijk et al. (1999
) showed
that the Ea for oxygen consumption of Antarctic eelpout
Pachycara brachycephalum was 99.4±5.9 kJ
mol1 versus 89.2±4.9 kJ
mol1 in congeneric temperate eelpout Zoarces
viviparus (acclimated to 12°C). After cold acclimation (at 4°C),
the rate of oxygen consumption of Z. viviparus was elevated, but
Ea had fallen to 55±3 kJ mol1
(Van Dijk et al., 1999
;
Zakhartsev et al., 2003
).
Sommer and Pörtner (2002
)
found that the Ea of NADP-dependent isocitrate
dehydrogenase of Arenicola marina was higher (83.3±10.6 kJ
mol1) in a cold-adapted White Sea population (sub-polar)
than in a temperate North Sea population (63.7±3.6 kJ
mol1). These examples suggest a variety of potential
responses by Ea to cold. Since coping with temperature
always involves compensatory strategies during short-term (acclimation) or
long-term (adaptation) temperature variation
(Clarke, 1991
), the observed
increase of Ea with a decrease in TA
raises the question of which mechanism of compensation was effective?
According to the literature, fish LDH at low acclimation temperature
undergoes molecular reorganization, which results in enhanced molecular
stability. LDH isolated from skeletal muscle of loach acclimated to 5°C (a
`cold' enzyme) has a higher thermal stability (heat resistance) and is more
resistant to urea-induced inactivation than LDH from fishes acclimated to
18°C (a `warm' enzyme; Klyachko et
al., 1995a; Ozernyuk et al.,
1994
). Accordingly, the enthalpy of denaturation
(
Hd) of enzyme purified from loach acclimated to
18°C was larger (23.3±1.6 J g1) than for the
enzyme purified from fish acclimated to 5°C (21.4±1.7 J
g1; Danilenko et al.,
1998
). The enthalpy of the unfolded state (Hu)
of both cold- and warm-acclimated enzymes should be the same since both
`forms' of the enzyme have identical amino acid sequences. The phenomenon of
Hd indicates that the enthalpy of the folded state
(Hf) of `cold' LDH increased and, as a consequence,
Hd was reduced
(
Hd=HuHf).
The specific heat capacity (Cp) measured at 25°C by
differential scanning microcalorimetry of cold LDH was higher
(1.39±0.03 J g1 K1) than that of
warm LDH (1.14±0.05 J g1 K1),
reflecting higher molecular stability in the cold
(Danilenko et al., 1998
). It
is clear that these differences relate to differences in the intramolecular
organization of the LDH extracted from fishes acclimated to different
temperatures. These differences are probably related to the number of
intramolecular interactions, which probably result in modified surface
properties of the molecule (Danilenko et
al., 1998
). Zasedateleva et al.
(1999
) found molecular
differences in the secondary structure of cold and warm LDH (acclimation at
5°C versus 18°C) of loach by means of circular dichroism
spectrometry. These differences become apparent during thermal denaturation of
the enzyme, which mainly relates to differences in the `melting' of
-helical structures: cold LDH exhibited a higher cooperativity in the
melting of
-helices and it occurred in a narrower temperature range
(7085°C) than in warm LDH (4085°C). However, the
temperatures of denaturation (Td=74°C) were
indistinguishable between the two acclimation forms of LDH at 5°C and
18°C (Danilenko et al.,
1998
; Klyachko et al.,
1995b
; Persikov et al.,
1999
). This is not in contrast to the other findings, because the
determination of Td is a relatively rough method to
estimate molecular differences.
It is well known that increasing the number of intramolecular hydrogen
bonds (Pace et al., 1996) as
well as non-polar bonds will stabilize protein structure and cause a higher
resistance to denaturation (Privalov and
Tsalkova, 1979
; Sowdhamini and
Balaram, 1993
). As a consequence, it increases the
Hf and Ea of the molecule. This
phenomenon is frequently observed in enzymes of microorganisms living in
thermal springs (Hochachka and Somero,
1984
). Therefore, one possible explanation for the observed
phenomenon of an increase in Ea of LDH with acclimation to
low temperature might be the introduction of additional weak interactions
within the LDH molecule. This additional weak interaction causes the observed
functional changes and enhanced molecular rigidity. This conclusion is
supported by the effect of urea, which exerts its effect by interfering with
weak bonds such as hydrogen bonds
(Ozernyuk et al., 1994
). It is
very likely that these additional hydrogen bonds were introduced in loop
regions, increasing their rigidity and consequently affecting the molecular
Ea and its catalytic efficiency. Zasedateleva et al.
(1999
) observed a change of
conformational structure of LDH during seasonal adaptation of loach and
related this shift to a change of electrostatic interactions between the
elements of secondary structure in LDH. Furthermore, Fields et al.
(2002
) studied homologous LDHs
from congeneric species of the goby fish Gillichthys seta and
Gillichthys mirabilis and proposed that subtle differences in
conformation around residual Tyr246, which is involved in subunit interaction
within the homotetramer and sits in a hinge between a static
-helix and
one involved in catalytic conformational changes, probably play a role both in
altered flexibility and in the potentially adaptive differences in kinetics
between the two LDH forms.
In fact, the analysis of activation enthalpy change
(Table 6) calculated for
corresponding acclimation temperatures revealed that in the condition of cold
acclimation H became higher by approximately 5.638.99
kJ mol1 (depending on the tissue). The magnitude of this
energy change is roughly the same as the enthalpy change involved in the
formation of one hydrogen bond (Haynie,
2001
), which is 5.70 kJ mol1 at 25°C. This
observation suggests that an additional 12 hydrogen bonds could appear
in the molecular structure of LDH under conditions of cold acclimation.
However, it is presently unclear how much change in activation enthalpy of the
enzymatic reaction is caused by the formation of one weak bond in enzyme
structure.
Our measurements indicate that LDH molecules did not undergo conformational phase-transition states within the range of assay temperatures (425°C). The Arrhenius plot was linear over the whole temperature range with no evident breakpoint, as shown by a high value of r2 (Table 6).
Recently, it was reported that the thermal stability of some enzymes can be
increased by calcium binding (Declerck et
al., 2000; Harris and
Davidson, 1994
). Danilenko et al.
(1998
) investigated this
possibility in cold and warm variants of LDH using x-ray fluorescence and did
not find any difference in the calcium contents of purified enzymes. Danilenko
et al. (1998
) also found no
phosphorus in both forms of LDH (cold and warm) from loach, indicating that
phosphorylation was not involved in the modification of thermostability. Some
low-molecular-mass osmolytes (free amino acids, glycine-based osmolytes) have
been found to increase the thermostability of enzymes
(Taneja and Ahmad, 1994
). Such
effects can be excluded for our enzyme preparations because of the use of
diluted homogenates. At the highest assay temperatures applied (30°C), the
final dilution factor of the tissue was 5x104 for muscle and
104 for liver. Therefore, it is very unlikely that
low-molecular-mass osmolytes were effective in the diluted solution.
All arguments mentioned above indicate a very high probability that
additional weak bonds were introduced into the molecular structure of LDH
adjusted to cold. Hochachka and Somero
(1984) pointed out that the
introduction of additional or enhanced weak interactions can considerably
increase the thermostability of a protein by stabilization of its
conformational state. This strategy may occur at the expense of a decrease in
catalytic efficiency. This argument is supported by the observed increase in
apparent Ea (Table
6) and the drop in Ksi. This indicates that an
increased enthalpy of the cold-adjusted enzyme is in line with a loss in
structural flexibility.
In summary, additional weak interactions within the quaternary structure of
the LDH may be one of the causes of the observed difference between LDHs from
cold- and warm-acclimated animals. A non-genetic post-translational
modification of the enzyme molecule probably leads to enhanced LDH folding
depending on acclimation temperature (temperature-driven protein folding). It
has been suggested previously that identical amino acid sequences (identical
primary structures) of LDHs from congeneric species of goby fish (Gobiidae)
can fold in different conformations
(Fields et al., 2002;
Fields and Somero, 1997
),
yielding conformational variants. Therefore, our data indicate that this
mechanism is not only involved on evolutionary time scales but probably
contributes to adaptation to seasonal temperature variation (acclimation).
Metabolic compensation
Comprehensive studies of thermal limitation indicate that the limits of
thermal tolerance are, in the first place, set at the level of complex
organismic processes and only secondarily at the molecular level (molecular or
structure phase transition, protein denaturation, etc.). Whole-animal aerobic
scope appears to be the first process limited at both low and high
temperatures (Frederich and Pörtner,
2000; Pörtner et al.,
1998
; Pörtner,
2002a
; Prosser,
1986
).
Aerobic and anaerobic pathways of metabolism are competing for pyruvate.
Therefore, the efficiency of energy production in particular tissues depends
on adequate channelling of this metabolite
(Somero, 1973). As was shown
earlier by Pörtner and Grieshaber
(Grieshaber et al., 1994
;
Pörtner and Grieshaber,
1993
) and later by Boutilier and St-Pierre
(2000
), the contributions of
aerobic and anaerobic pathways to total energy production depend on oxygen
availability to mitochondria. Below the critical oxygen tension
(Pc) the contribution of anaerobic pathways rises. Thus,
survival in constantly fluctuating environments depends on the relationship
between aerobic and anaerobic pathways and their capacity for ATP
production.
The net increase in the apparent Eas for pyruvate
reduction by LDH during acclimation to cold temperatures, combined with an
increase in aerobic capacity (Lannig et
al., 2003) and the associated increase of mitochondrial density,
may be one mechanism of metabolic control and indicates a downregulation of
anaerobic capacity when the relative fraction of aerobic metabolism increases.
This might help to avoid pyruvate limitation for aerobic oxidation
(maintenance of aerobic scope) and thereby increases the efficiency of aerobic
metabolism at low temperature. Other features involved in the change of LDH
functional properties include a reduced range of pyruvate concentrations
required for the maximal capacity of LDH
(
=
).
Although the adjustment of mitochondrial densities and their kinetic
properties are one of the key mechanisms to regulate metabolic capacities
during acclimation to low temperature, the outlined mechanism
(temperature-driven enzyme folding) contributes to create favourable
conditions for the aerobic functioning of mitochondria at low temperature.
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Acknowledgments |
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