First published online January 31, 2006
Journal of Experimental Biology 209, 677-688 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02052
Suppression of Na+/K+-ATPase activity during estivation in the land snail Otala lactea
Christopher J. Ramnanan and
Kenneth B. Storey*
Institute of Biochemistry and Department of Biology, College of
Natural Sciences, Carleton University, 1125 Colonel By Drive, Ottawa, Ontario,
Canada K1S 5B6
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Fig. 1. Na+/K+-ATPase activity in hepatopancreas from active
and estivated O. lactea as a function of assay pH. Assays were
conducted under optimal substrate concentrations at 22°C. Data are means
± s.e.m., N=4 independent preparations of enzyme.
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Fig. 2. Na+/K+-ATPase activity in O. lactea (A) foot
muscle (mean ± s.e.m., N=9) and (B) hepatopancreas (mean
± s.e.m., N=4) as a function of Mg.ATP concentration.
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Fig. 3. Substrate affinity for ATP as a function of temperature for
Na+/K+-ATPase activity in O. lactea in (A) foot
muscle and (B) hepatopancreas. Values are means ± s.e.m., N=4
independent preparations of enzyme except for foot muscle control and
estivated activity, which are N=9. *Significantly
different from the corresponding control incubation; Student's
t-test, P<0.01.
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Fig. 4. Effect of in vitro incubations to stimulate the activities of
endogenous (A) protein kinases and (B) protein phosphatases on
Na+/K+-ATPase activity in foot muscle extracts from
active and 10-day estivated O. lactea. Incubation time was 4 h prior
to assay under Vmax conditions at 22°C. Values are
means ± s.e.m., N=4 independent determinations.
*Significantly different from the corresponding control incubation;
Student's t-test, P<0.01;
 P<0.05. For details of incubation conditions,
see Materials and methods.
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Fig. 5. Effect of in vitro incubations stimulating the activities of
endogenous (A) protein kinases and (B) protein phosphatases on
Na+/K+-ATPase activity in hepatopancreas extracts from
active and 10-day estivated O. lactea. Other information as in
Fig. 4.
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Fig. 6. Effect of calcium concentration on the stimulation by endogenous protein
kinases of Na+/K+-ATPase activity in foot muscle
extracts from active snails. In vitro incubations stimulated PKA, PKC
or PKG and the resulting change in Na+/K+-ATPase
activity is expressed as a percentage of the activity in control incubations
where protein kinases were not stimulated. Other information as in
Fig. 4.
*Significantly different from the corresponding incubation with
zero calcium concentration; Student's t-test, P<0.01.
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Fig. 7. Arrhenius plots showing Na+/K+-ATPase maximal
activity as a function of temperature (Kelvin) for (A) foot muscle and (B)
hepatopancreas from O. lactea. Values are means ± s.e.m.,
N=4 independent preparations of enzyme. Assay mixtures were
pre-incubated for 5 min in water baths set to specified temperatures prior to
initiating reactions by the addition of Mg2+.ATP. Assays were
conducted at maximal substrate concentrations. Arrhenius activation energy
(EA) was calculated from the linear part of the
graphs.
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Fig. 8. Western blots showing Na+/K+-ATPase protein content
in (A) foot muscle and (B) hepatopancreas of active and 10-day estivated
snails. Representative blots are shown for four independent samples. Band
densities were normalized against the combined measured densities of three
protein bands stained strongly with Coomassie Blue that did not change between
control and estivated states, and histograms show the mean normalized band
densities (± s.e.m., N=10 for foot muscle, N=8 for
hepatopancreas). The band shown corresponds to  100 kDa, as determined
from a standard curve developed using Kaleidoscope protein size markers, and
is consistent with the expected alpha subunit size of
Na+/K+-ATPase.
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Fig. 9. Structural unfolding and enzymatic inhibition by urea of foot muscle
Na+/K+-ATPase from active and estivating snails. Enzyme
extracts were denatured overnight with different concentrations of urea and
then either treated with thermolysin or assayed under optimal conditions. (A)
Mean relative Na+/K+-ATPase protein remaining (as
quantified from band intensities on western blots) after overnight incubation
with urea and subsequent proteolysis with thermolysin; values are means
± s.e.m., N=3 independent trials. (B) Mean relative activity
of foot muscle Na+/K+-ATPase from active and estivating
snails as a function of overnight incubation with different concentrations of
urea; values are means ± s.e.m., N=3.
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© The Company of Biologists Ltd 2006
