Osmotic effects on arginine kinase function in living muscle of the blue crab Callinectes sapidus
Department of Biological Sciences, University of North Carolina at
Wilmington, 601 South College Road, Wilmington, NC 28403-3297, USA
* Present address: Department of Biology, University of Vermont, Burlington, VT
05405, USA
Author for correspondence (e-mail:
kinseys{at}uncwil.edu
)
Accepted 8 April 2002
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Summary |
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Key words: arginine kinase, osmoregulation, haemolymph, muscle, blue crab, Callinectes sapidus, crustacean, nuclear magnetic resonance, salinity
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Introduction |
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Although the blue crab is a capable osmoregulator at low salinities,
hemolymph osmolarity may still fluctuate from approximately 600 to 1000
mosmoll-1 as the environmental salinity changes from 0 to
35, respectively (Lynch et al.,
1973
; Mantel and Farmer,
1983
; Piller et al.,
1995
). Since the tissues remain iso-osmotic with the hemolymph,
the intracellular osmolyte composition and concentrations may change
dramatically when the animal experiences a change in environmental salinity
(Gérard and Gilles,
1972
; Gilles,
1979
). One way in which the tissues of blue crabs, like those of
other euryhaline organisms, compensate for salinity challenges is by altering
the concentrations of free amino acids and of other compatible or
counteracting solutes to maintain cells in an iso-osmotic condition
(Gérard and Gilles,
1972
). These compounds help to maintain proper cell volume without
the destabilizing effects on protein structure that are typically induced by
inorganic ions (Brown and Simpson,
1972
; Clark and Zounes,
1977
; Bowlus and Somero,
1979
; Yancey et al.,
1982
; Pierce,
1982
). A large body of in vitro studies conducted by
these and other authors have detailed the nature of the perturbing effects of
inorganic ions on enzymes and some of the mechanisms by which compatible and
counteracting solutes preserve enzyme function (for reviews, see Yancey,
1994
,
2001
). More recently, it has
been demonstrated that cells made hypertonic with elevated levels of NaCl
experience double-strand breaks in DNA
(Kültz and Chakravarty,
2001
) and induce heat-shock proteins
(Petronini et al., 1993
;
Sheikh-Hamad et al., 1994
).
These studies underscore the protective effects of organic osmolytes, since
cells made hypertonic with the stabilizing solute urea did not undergo DNA
degradation (Kültz and Chakravarty,
2001
), and heat-shock protein induction was inhibited by the
presence of the compatible solute betaine
(Petronini et al., 1993
;
Sheikh-Hamad et al.,
1994
).
Despite a sizable literature describing the effects of solutes on proteins,
the consequences of salinity challenges and the associated alterations in the
intracellular environment on enzyme function in intact organisms or tissues
are largely unknown. Osmotic stress may be particularly disruptive to cell
function during rapid changes in salinity since the compensatory adjustments
of intracellular organic solute concentrations often occur over a period of
hours or days, lagging behind the onset of changes in hemolymph osmolarity
(Dall, 1975;
Bartberger and Pierce, 1976
;
Gilles, 1979
;
Pierce, 1982
). During this
transient period of acclimation, inorganic ions that perturb enzyme function
may be the principal intracellular osmolytes involved in cell volume
regulation (Warren and Pierce,
1982
).
Arginine kinase (AK) is a member of the phosphagen kinase family that
catalyzes the reversible transfer of a high-energy phosphate from the
phosphagen arginine phosphate to ADP to form ATP:
![]() | (1) |
AK has been the subject of more in vivo kinetic analyses than any
other phosphagen kinase except creatine kinase, which is the functional analog
to AK found in vertebrates and some invertebrates. Magnetization transfer NMR
has been employed to measure steady-state reaction flux in the phasic adductor
muscle of the scallop Argopecten irradians
(Graham et al., 1986) and the
abdominal muscle of the crayfish Orconectes virilis
(Butler et al., 1985
). The
in vivo temperature-dependence of AK has been examined in leg muscle
of the crab Carcinus maenas
(Briggs et al., 1985
) and in
abdominal muscle of the shrimp Sycionia ingentis
(Fan et al., 1992
). More
recently, magnetization transfer was used to assess the effects of
pentachlorophenol and hypoxia on the rates of AK flux in red abalone
Haliotis rufrescens (Shofer et
al., 1996
). However, to our knowledge, there is no information
regarding intracellular AK function related to physiologically relevant
variations in environmental salinity.
The present study used 31P-NMR saturation transfer to examine AK flux in isolated blue crab muscle in osmotic steady state and under hyperosmotic and hypo-osmotic conditions. AK flux varied by nearly twofold across the entire range of osmotic conditions examined, although the enzyme appeared to be largely unaffected by moderate osmotic challenges.
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Materials and methods |
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Arginine phosphate metabolite assays
Arginine phosphate concentrations in muscle were measured
spectrophotometrically for animals that had been exposed to each of the
acclimation salinities. The concentrations of ATP and Pi could then
be determined from their NMR peak area relative to the peak area of arginine
phosphate (see below). Between 200 and 500 mg of dark levator muscle was
homogenized using a Fisher Powergen 125 homogenizer in 2.5 ml of ice-cold 7%
HClO4 containing 1 mmoll-1 EDTA. The sample was
centrifuged for 10 min at 9880g in an Eppendorf 5415C microcentrifuge
at 4°C. The supernatant was titrated to pH6.5 using 3 moll-1
KOH containing 50 mmoll-1 Pipes and allowed to sit on ice for 10
min. The sample was centrifuged again at 9880g for 15 min at 4°C.
The concentrations were measured via an enzymatically linked assay.
The AK reaction was coupled to the production of NADPH, which was monitored at
a wavelength of 340 nm on a Pharmacia Ultrospec 4000 spectrophotometer. The
assay medium contained 30 mmoll-1 Tris/HCl, 2.5 mmoll-1
MgCl2, 2.5 mmoll-1 D-glucose, 0.63 mmoll-1
NADP, 150 units of glucose-6-phosphate dehydrogenase and the muscle extract.
Hexokinase (3 units) was then added, and the absorbance was allowed to
stabilize following the consumption of endogenous ATP. This was followed by
the addition of 0.5 mmoll-1 ADP and 5 units of arginine kinase; the
observed change in absorbance was proportional to the concentration of
arginine phosphate.
Measurement of arginine kinase flux
Swimming leg dark levator muscle was excised and immediately transferred to
a Petri dish containing blue crab saline solution gassed with a mixture of
99.5% O2/0.5% CO2. The white muscle was mechanically
stripped to isolate the dark levator from surrounding muscle. The superfusion
medium meant to mimic that of animals exposed to a salinity of 35 had
an osmolarity of 960mosmoll-1 and consisted of
470mmoll-1 NaCl, 8mmoll-1 KCl, 15mmoll-1
CaCl2, 10mmoll-1 MgSO4 and
10mmoll-1 Hepes at pH7.4 (Tse
et al., 1983
). The solutions for animals maintained at the more
dilute salinities of 17 and 5
were prepared using the same ionic
composition as described above with the addition of deionized water to dilute
the saline solution to 720 and 640mosmoll-1, respectively.
Osmolarity was measured using a VAPRO 5520 vapor-pressure osmometer.
The isolated muscle was tied at resting length to a plastic capillary tube
using 3-0 surgical suture. The muscle was then placed in a 10mm diameter NMR
tube and connected to a superfusion system with peristaltic pumps that
continuously washed the tissue with oxygenated saline solution at a flow rate
of 10mlmin-1. The temperature was maintained at 20°C with a
1016S Isotemp recirculating water bath. The muscle preparation and superfusion
tubing were lowered into the NMR magnet where the sample was `mated' with a
10mm NMR probe. Experiments were designed to simulate the ionic environment
experienced by the muscle in vivo during a rapid change in
environmental salinity. AK flux was therefore measured under control,
hypo-osmotic and hyperosmotic conditions
(Table 1). For example, a
muscle from a control animal was superfused with a medium equivalent to the
osmolarity existing in the animal's blood after the 7-day exposure (e.g. an
animal exposed to a 5 environmental salinity and the muscle
preparation exposed to a superfusion medium of 640mosmoll-1;
Piller et al., 1995
). In
contrast, a hyperosmotic shock treatment involved superfusing the muscle in a
saline solution with an osmolarity greater than that existing in the animal's
blood after the 7-day exposure period (e.g. an animal exposed to a 5
environmental salinity and the muscle preparation exposed to a superfusion
medium of 960mosmoll-1). This example would simulate a rapid move
from a salinity of 5 to 35
.
|
NMR spectra were obtained using a Bruker 400MHz DMX spectrometer housed in the Department of Chemistry at the University of North Carolina at Wilmington. The sample was tuned to the phosphorus precessional frequency of 162MHz and shimmed on the residual proton signal arising from water to optimize magnetic field homogeneity. Initial spectra were obtained to ensure tissue viability. These spectra were acquired using a 45° (25µs) excitation pulse and a 1 s relaxation delay. Ninety scans were acquired, and 25Hz exponential line-broadening was applied before Fourier transformation.
To calculate AK flux from the saturation transfer data, it is necessary to
determine the actual concentrations of arginine phosphate and ATP at the time
the NMR measurement is made. As stated above, we directly measured the
arginine phosphate levels spectrophotometrically (and we inferred the amount
of ATP from the NMR spectrum) at the three acclimation salinities. However,
changing the osmolarity of the superfusion medium may lead to energetic
challenges to the tissue that result in changes in the concentrations of
arginine phosphate and ATP. Therefore, for a subset of hyperosmotic and
hypoosmotic treatment groups, a time series of spectra was acquired to assess
new steady-state concentrations of metabolites and ensure stable levels of
high-energy phosphates during the experiment. The same parameters were used as
previously described for initial spectra. In addition, the intracellular pH
(pHi) of the muscle preparation was determined in each experiment from the
chemical shift of the Pi peak relative to that of arginine
phosphate (Kinsey and Moerland,
1999).
The pseudo-first-order unidirectional rate constants in the forward and
reverse directions (kforward and
kreverse) were measured for the AK reaction using a
saturation transfer method (Briggs et al.,
1985; Graham et al.,
1986
). A 10s low-power pulse was used to saturate selectively
either the resonance for arginine phosphate or that for
-ATP. This was
followed immediately by a 90° (45µs) broadband excitation pulse that
allowed for signal detection. One hundred and twenty-four scans were averaged,
so that each spectrum was collected in 21 min. To measure the forward
reaction, the
-ATP resonance was saturated; for the reverse reaction,
the arginine phosphate resonance was saturated. These spectra were compared
with control spectra in which no peak was saturated. Here, the saturating
pulse was applied at the opposite side of the unsaturated peak at the same
frequency offset as in the corresponding saturated spectrum. This controls for
possible partial saturation of the unsaturated peak during selective
irradiation. The total experimental time was 84 min, which included the two
saturated and the two control spectra. Peaks were integrated to determine the
area under each peak using Bruker X-Win NMR software. The rate constant
k was calculated from the expression:
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Arginine kinase activity assays
Maximal AK activity was measured spectrophotometrically to determine its
salinity-dependence and to compare in vitro activity with in
vivo flux. It was assumed that, during the 84min experiments, there would
be no significant osmolarity-induced changes in the total amount of AK, so
activity was measured for the control animals only. We believe that this
assumption is valid because, to our knowledge, there is no evidence of AK
induction by salinity or any other environmental variable in crustacean
muscle. This is probably because the principal function of AK in crustacean
muscle is temporal ATP buffering (see Discussion), and upregulation of the
enzyme would not enhance this function. Further, AK occurs in very high
concentration in crustacean muscle, so if protein turnover in general were
affected by the experimental treatments, the percentage change in AK
concentration is likely to be relatively small.
The tissue was homogenized in 9vols of 100mmoll-1 glycine and 10mmoll-1 ß-mercaptoethanol (pH8.6). The homogenate was centrifuged in a Beckman J2-21M/E for 10min at 12000g at 4°C. Assays were performed using a Pharmacia Ultrospec 4000 spectrophotometer with the temperature maintained at 20°C. The activity was measured by enzymatically linking the AK reaction to the oxidation of NADH, which was monitored at a wavelength of 340nm. The assay medium was 65mmoll-1 Tris/HCl (pH8.0), 38 mmoll-1 KCl, 13mmoll-1 magnesium acetate, 5mmoll-1 ATP, 1.25mmoll-1 phosphoenolpyruvate, 0.25mmoll-1 NADH and excess pyruvate kinase/lactate dehydrogenase. Basal ATPase activity was initiated by the addition of the extract. After a steady-state trace had been recorded, 10mmoll-1 arginine was added to the mixture to initiate arginine kinase activity.
1H-NMR
1H-NMR was used to measure the relative concentration of organic
osmolytes in muscle tissue under each experimental condition. Tissue was
prepared following the same time course of osmolarity exposure as in the
saturation transfer experiments. Since the 1H-NMR spectra could be
collected in several minutes, the samples were not superfused. The sample was
placed in a 5mm NMR tube, shimmed to the proton signal arising from water and
tuned to 400MHz. The water resonance was suppressed using a 5s presaturation
pulse. The excitation pulse was 11µs, the relaxation delay was 5s and 128
scans were acquired. The total experiment time was 12min. A 0.50Hz exponential
line-broadening function was applied before Fourier transformation.
Statistical analyses
Normality of data was determined using a 2 goodness-of-fit
test, and homogeneity of variances was assessed using Bartlett's test. Two-way
analysis of variance (ANOVA) was used to test all NMR-derived data for
significant effects of acclimation salinity, experimental osmolarity of the
medium and for interaction of these two parameters. One-way ANOVA was used to
test for significant effects of acclimation salinity for NMR-derived data from
control animals and for spectrophotometrically measured arginine phosphate
concentrations and AK activities. Student's t-tests were used for
pairwise comparisons. Linear regression analysis was used to assess the
dependence of AK rate constants and flux rates on the extent of osmotic
stress. The level of significance for analysis was P<0.05, and the
data are reported as means±S.E.M. All data were analyzed using SAS-JMP
statistical software version 4.04 (SAS Institute, Cary, NC, USA).
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Results |
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Table 2 includes the
concentrations of arginine phosphate, -ATP and Pi for the
control treatments, determined spectrophotometrically (arginine phosphate) and
using NMR (
-ATP, Pi). In addition, concentrations are
presented for the hypo- and hyperosmotic treatments, which were derived
exclusively from the NMR data using the control concentrations of arginine
phosphate, to illustrate the stability of the concentrations of high-energy
phosphate compounds (Table 2).
One-way ANOVA indicated that muscle arginine phosphate concentration varied
significantly across the experimental salinities from 11.9 µmol
g-1 wet mass for animals acclimated to 17
to 17 µmol
g-1 wet mass for animals acclimated to 35
. Animals from
5
salinity had intermediate muscle arginine phosphate concentrations.
Two-way ANOVA was used to test for an effect of acclimation salinity,
experimental osmolarity or an interaction of the two on the other high-energy
phosphate concentrations. There were no significant differences in the levels
of
-ATP or Pi or in the arginine phosphate/
-ATP or
-ATP/Pi concentration ratios across either acclimation
salinity or experimental osmolarity. Further, acclimation salinity or
experimental osmolarity did not significantly affect pHi
(Table 2).
|
Arginine kinase fluxes
The exchange of the high-energy phosphate catalyzed by AK is apparent from
the reduction in amplitude of the arginine phosphate resonance when the
-ATP peak is saturated, as illustrated in the representative spectra
presented in Fig. 2. The
forward (Kforward) and reverse
(Kreverse) pseudo-first-order unidirectional rate
constants and the corresponding forward and reverse fluxes for control
treatments are presented in Table
3. Among the control animals only, a one-way ANOVA indicated that
there was no significant effect of acclimation salinity on rate constants or
flux measurements. When the control, hypo-osmotic and hyperosmotic data were
analyzed together, two-way ANOVA indicated a significant effect of acclimation
salinity for Kreverse, forward flux and reverse flux. This
analysis revealed no significant effect of experimental osmolarity.
|
|
However, careful inspection of the data did reveal a general pattern in which mean values for rate constants and flux rates were relatively high for the hypo-osmotic treatments and relatively low for the hyperosmotic treatments (Table 3). This pattern is more clearly observed by plotting the rate constants and fluxes against the difference in experimental osmolarity from the control value (Fig. 3). The most hyperosmotic treatments can be seen to have rate constants and flux rates that are approximately 1.7 times lower than those of the most hypo-osmotic treatments. Linear regressions of both rate constants and both fluxes against the difference from control osmolarity were significant in all cases except for Kforward (Fig. 3). The ratio of forward flux to reverse flux (F/R) ranged from 0.81 to 1.08 and had a grand mean of 0.98±0.07 (N=37), which is very close to the expected value of 1 for an enzyme-catalyzed reaction at equilibrium (Table 3).
|
Arginine kinase activity assays
In vitro maximal AK activities (measured in the forward direction)
were approximately 10 times greater than the forward flux values determined in
the muscle tissue (Table 3).
Maximal AK activity was also higher in animals acclimated to more dilute media
than in animals from full-strength sea water. Animals acclimated to a salinity
of 35 had the lowest AK activity, while animals acclimated to a
salinity of 17
had the highest AK activity. Interestingly, the maximal
AK activity was inversely proportional to the arginine phosphate concentration
(Tables 2,
3).
1H-NMR
A dominant peak that demonstrated salinity-induced variation was observed
at a chemical shift of 3.2p.p.m. in 1H-spectra
(Fig. 4). We cannot attribute
this large resonance to any of the amino acids that have been shown to be
important osmolytes in blue crab muscle
(Gérard and Gilles,
1972), although the triplet CH2 peak of arginine
probably contributes slightly to the large peak at 3.2p.p.m. The peak has a
chemical shift consistent with a methylamine compound such as betaine
(including the presence of the small peak at 3.85p.p.m.;
Fig. 4), and NMR spectra
collected from a muscle extract before and after it was spiked with betaine
support this conclusion. To our knowledge, betaine has not previously been
reported to occur in abundance in blue crab muscle, but it is an important
osmolyte in the euryhaline crab Eriocheir sinensis
(Bricteux-Grégoire et al.,
1962
). Despite some uncertainty in the assignment of this peak, we
believe that it represents an important organic osmolyte on the basis that its
concentration is both high and sensitive to salinity. The relative
concentration of this resonance was determined by normalizing the peak area to
the cumulative area of all other proton peaks, excluding that of water.
Although some of the other small resonances visible in the
1H-spectra may also be derived from regulated osmolytes, the peaks
were too small and broad to estimate concentration changes reliably in the
living muscle preparation.
|
Two-way ANOVA of the 1H-NMR results indicated that the
concentration of betaine changed significantly as a function of acclimation
salinity but not as a function of experimental osmolarity
(Fig. 5). As expected, the
concentration of this osmolyte was highest in muscle from animals acclimated
to 35 and lowest in animals acclimated to 5
. The mean values
were approximately 40% lower in muscle from animals acclimated to 5
than for those acclimated to 35
. Therefore, the 7-day salinity
acclimation period induced changes in the concentration of this osmolyte (and
probably others), but during the relatively short time course of the
saturation transfer experiments the concentration was unaltered.
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Discussion |
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The present study found that the levels of the high-energy phosphate
compounds (Fig. 1) and pHi were
not altered by hypo- or hyperosmotic treatments. The stability of the levels
of arginine phosphate, ATP and Pi during even the most extreme
treatments implies that there is not a substantial energetic cost in terms of
whole-muscle ATP demand during osmotic stress. A sizable increase in ATP
demand would be manifested as decreased [ATP]/[Pi] and [arginine
phosphate]/[ATP] ratios (for reviews, see
van den Thillart and van Waarde,
1996; Wasser et al.,
1996
). However, we cannot rule out the possibility that there may
have been an undetectable localized perturbation to high-energy phosphate
concentrations in regions near the sarcolemmal membrane, where regulatory ion
pumps may be active (Combs and Ellington,
1997
).
In vitro AK activities and arginine phosphate concentrations were
consistent with values found from oxidative tissues from other invertebrates
(Tables 2,
3) (for a review, see
Ellington, 2001). Both AK
activities and arginine phosphate concentrations were different for each
acclimation salinity, but the differences were not proportional to salinity
(i.e. the lowest-salinity treatment, 5
, had intermediate values for
both measurements). It is interesting that the values for AK activity and
arginine phosphate levels were inversely related at the three acclimation
salinities, although we do not know the biological significance of this
relationship. In vitro AK activity for the forward reaction was
approximately 10 times higher than forward flux measured in intact muscle
(Table 3). This discrepancy has
been reported previously for AK in magnetization transfer experiments
(Briggs et al., 1985
;
Platzer et al., 1999
). It has
been suggested that the extraction procedure for the in vitro assays
may liberate compartmentalized AK that has a lower activity in resting muscle
(Briggs et al., 1985
). In
contrast, Graham et al. (1986
)
found that the phasic adductor muscle in scallop had flux values that were in
accord with in vitro maximal activity values.
Enzyme F/R flux ratios measured using NMR magnetization transfer
methods have frequently been found to deviate from a value of 1, and this
phenomenon has been observed for AK. In muscle from the shore crab
Carcinus maenas, F/R ratios were nearly 2, and Briggs et al.
(1985) suggested that the cause
was either metabolic compartmentation or multi-site exchange that led to an
underestimation of reverse flux. In the present study, the AK F/R
flux ratios in muscle were very close to 1, with a grand mean of
0.98±0.07 (Table 3). An
F/R ratio of 1 would be expected for an enzyme near equilibrium, and
our results are similar to that found in scallop phasic adductor muscle
(Graham et al., 1986
).
The major finding of the present study is that AK flux in muscle varies over a 1.7-fold range under different osmotic conditions (Table 3; Fig. 3). The results suggest an effect of both acclimation salinity and experimental osmolarity; however, only the former effect was significant in the two-way ANOVA. It should be reiterated that one-way ANOVA of the three control treatments alone did not indicate a significant effect of acclimation salinity. The similarity of the control values indicates that AK function is independent of salinity in crabs that have been exposed to a given salinity long enough to reach a new `acclimated' osmotic steady state. However, the significant acclimation salinity effect found across all treatments suggests that salinity history may play a role in the response of AK to hypo- or hypersmotic conditions.
The rate constants and fluxes were plotted as a linear function of the extent of osmotic stress, with the highest values observed in the most hypo-osmotic treatments and the lowest values found in the most hyperosmotic treatments (Fig. 3). Linear regression was used here for statistical convenience and because a reasonably good fit was attained, but we do not mean to imply that this relationship is truly linear over a broad osmotic range. Although the mechanism by which AK flux was altered in our experiments is not known, the systematic manner in which flux was affected by the extracellular osmotic conditions may offer some insight into possible causes. Three scenarios that might lead to a change in the AK flux measurements include (i) a change in the muscle energetic state, (ii) a change in cell volume that would alter substrate concentrations and (iii) a direct effect of inorganic ions and/or organic osmolytes on AK function.
Since ATP, ADP and arginine phosphate are all substrates for the AK
reaction, changes in the energetic state of the cell associated with osmotic
stress might be expected to alter AK flux. As stated above, however, our hypo-
and hyperosmotic treatments do not appear to be an energetic challenge to the
muscle as a whole on the basis of the stability of the levels of high-energy
phosphate compounds (Fig. 1;
Table 2). A second reason why
energetic changes should not affect AK flux is that the enzyme has a high
activity in the dark levator muscle (Table
3), and it is likely that the major functional role of AK in this
tissue is temporal ATP buffering. This assumption is based on the fact that,
while AK is localized to mitochondria in tissues from arthropods
(Ellington, 2001), the vast
majority of activity is associated with the cytosolic form of AK
(Doumen and Ellington, 1990
).
Furthermore, mitochondrial AK does not appear to be coupled functionally to
oxidative phosphorylation in the manner of mitochondrial creatine kinase so
that AK flux does not respond directly to ATP demand
(Doumen and Ellington, 1990
).
The available evidence therefore suggests that AK is a simple equilibrium
enzyme, and that the flux rate should not be susceptible to moderate changes
in energetic demand. A similar argument might be made regarding the
concentration of arginine, which is both a substrate for the AK reaction and a
salinity-sensitive osmolyte in blue crab muscle
(Gérard and Gilles,
1972
). However, the Km of AK for arginine is
typically less than 1 mmol l-1
(Platzer et al., 1999
;
Suzuki et al., 2000
), which is
well below the concentration of arginine supported by the AK reaction at
equilibrium (Teague and Dobson,
1999
). Nevertheless, to establish definitively the envelope of
expected flux values on the basis of in vivo metabolite
concentrations would require a detailed kinetic analysis similar to those
offered for creatine kinase by McFarland et al.
(1994
) and van Dorsten et al.
(1997
).
Although the levels of high-energy phosphate compounds remained constant in
the NMR measurements (Fig. 1),
changes in cell volume associated with the hypo- and hyperosmotic treatments
would alter the cellular concentrations of AK substrates. The most extreme
treatments in the present study simulated a change in the hemolymph osmolarity
of approximately 300 mosmol l-1. Lang and Gainer
(1969) examined cell volume
regulation in blue crab muscle using osmotic treatments of the same magnitude
as our most extreme tests. It was found that acute hyperosmotic shock led to
an immediate reduction in cell size to 80-85% of the initial cell volume, and
this reduced volume was maintained for at least 5h. Hypo-osmotic treatments
led to an immediate and larger increase in cell size, but was followed by a
fairly rapid (1 h) adjustment of cell volume to approximately 110% of the
initial volume. Cells were then maintained for at least several hours at this
partially corrected volume (Lang and
Gainer, 1969
).
If similar cell volume changes occurred in our study, then this would lead
to a dampening of the osmotic effects on AK flux demonstrated in
Fig. 3. Hypo-osmotic treatments
would lead to swelling and a reduction in substrate concentration, which would
lead to reduced flux. In contrast, cell shrinking under hyperosmotic
treatments would increase flux. The rate constants would not be affected by
volume changes. If we assume volume changes comparable with that described
above and adjust the levels of arginine phosphate and -ATP accordingly,
the pattern observed in Fig. 3
remains the same. However, the difference in flux between the extreme hypo-
and hyperosmotic treatments is reduced by approximately 50%. If these
volume-adjusted data are re-analyzed in a two-way ANOVA, there is still a
significant effect of acclimation salinity for the forward flux measurements
as described above, but this effect is no longer significant for the reverse
flux. Although this conservative analysis represents an overestimate of the
effects of volume change on most of our data, it indicates that cell volume
changes are probably responsible in part for the patterns observed in
Fig. 3.
It is also likely that changes in the composition of the intracellular
environment contribute to the observed osmotic effects on AK function. In
their analysis of blue crab muscle, Lang and Gainer
(1969) found that inorganic
ion concentrations could not be adequately explained by volume changes alone.
Intracellular K+ concentration was particularly variable, and its
concentration decreased from 187 to 105 mmol l-1 following
hypo-osmotic incubation. Gérard and Gilles
(1972
) measured intracellular
concentrations of Na+, K+ and Cl- in C.
sapidus muscle during hypo-osmotic shocks comparable with our
intermediate treatments and found that these three ions showed a cumulative
decrease of 55 mosmol kg-1 intracellular water at the lower
salinity. A number of studies have shown that changes in the concentrations of
Na+, K+ and Cl- over similar ranges may lead
to moderate or even dramatic effects on enzyme catalytic capacity in
vitro (Bowlus and Somero,
1979
; Yancey et al.,
1982
; Yancey,
1994
). In the present study, our hyperosmotic treatments tended to
reduce flux (Table 3;
Fig. 3). Under these
conditions, AK would be exposed to relatively high concentrations of inorganic
ions, which may disrupt protein function, and relatively low concentrations of
organic osmolytes, which may help stabilize protein structure
(Yancey et al., 1982
; Yancey,
1994
,
2001
). In contrast,
hypo-osmotic treatments tended to yield higher AK fluxes
(Table 3;
Fig. 3). Here, the compatible
solute concentration is presumably high and the inorganic ion concentration is
relatively low.
The 1H-NMR data suggest that this interpretation of the organic
osmolyte concentrations in our treatments is accurate. The peak tentatively
assigned to betaine changed in amplitude with acclimation salinity as expected
(Fig. 5), but it did not change
during the time course of our hypo- or hyperosmotic treatments. Lacking a
complete organic osmolyte response, it appears that, for cell volume
regulation purposes (or as a result of cell volume changes), concentrations of
potentially perturbing inorganic ion were relatively high during the shortterm
hyperosmotic treatments and relatively low during the hypo-osmotic treatments
(Table 1;
Pierce, 1982).
An interesting alternative hypothesis is that planar anions, such as
Cl-, may inhibit the enzyme in vivo by stabilizing the
abortive dead-end complex, enzyme·MgADP·arginine. This effect
has been demonstrated for the creatine kinase reaction in vivo
(McFarland et al., 1994), and
it could be argued that AK is similarly inhibited under hyperosmotic
conditions, while inhibition is relieved in the hypo-osmotic treatments.
However, the anion stabilization of the dead-end complex is less pronounced in
AK than in creatine kinase, and the most likely anion candidate with respect
to osmoregulation, Cl-, has to our knowledge not been shown to
stabilize the AK complex in vitro (Anosike and Watts, 1976). We
therefore conclude that the non-specific ionic effects described above are a
more likely modulator of enzymatic flux in blue crab muscle.
The observed changes in AK flux raise a question as to the physiological
relevance of salinity-induced changes in the osmotic state of blue crab
muscle. Our hypo- and hyperosmotic treatments simulated rapid changes in
environmental salinity that temporarily disrupted the balance between levels
of inorganic ions and organic osmolytes found in the acclimated state. While
small estuaries can undergo dramatic shifts in salinity over a short period,
the typical daily variation in salinity is likely to be less than that
simulated in our most extreme osmotic challenges. Also, in using isolated
muscle preparations, we have removed the gill osmoregulatory machinery that
may serve as a temporal buffer of the osmolarity of the hemolymph. Even if the
environmental salinity is changed instantaneously, blue crab hemolymph
osmolarity may change more gradually because of the ion-transporting capacity
of the gills (Gilles, 1979;
Towle et al., 1994
).
Therefore, our most extreme treatments may represent an osmotic condition that
exceeds that routinely experienced in nature. In fact, the range of AK flux is
quite small across the more modest osmotic treatments used in this study
(Table 3; Fig. 3). In this light, the
changes in AK flux that we observed should probably be considered to be fairly
modest, perhaps even indicating a remarkable preservation of AK function
during dramatic changes in the extracellular environment.
AK was selected as a model enzyme for examining the effect of osmotic
challenges on enzyme function because it is a tractable system using
non-invasive magnetization transfer methods. However, AK may not be an ideal
enzyme for this purpose. The bulk of AK in arthropod muscle cells is thought
to be a soluble monomeric protein
(Ellington, 2001). However,
because of the disruptive effects of inorganic ions on proteinprotein
interactions (Yancey et al.,
1982
), multimeric enzymes may be more susceptible to intracellular
osmotic perturbations. Therefore, non-invasive measurements of enzymes that
are composed of multiple subunits and of those that are functionally localized
to specific regions within the cell would be beneficial.
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