From the Department of Biological Chemistry, Weizmann Institute of
Science, Rehovot 76100, Israel
This work provides evidence for interactions
between fragments of "19-kDa membranes," a trypsinized preparation
of Na,K-ATPase that retains cation occlusion and ouabain binding.
Previously, we reported rapid thermal inactivation of
Rb+ occlusion at 37 °C (Or, E., David, P.,
Shainskaya, A., Tal, D. M., and Karlish, S. J. D. (1993)
J. Biol. Chem. 268, 16929-16937). We describe here
the detailed kinetics of thermal inactivation. In the range
25-35 °C, a two-step model (N
U
I, where N is the native
species, U is the reversibly unfolded intermediate, and I is the
irreversibly denatured form) fits the data. Reversibility of
inactivation has been observed at 25 °C, consistent with the model.
At 37 °C and higher temperatures, the data can be fitted to the
simple mechanism N
I, i.e. U is not significant as an intermediate. Occluded cations (Na+, Rb+,
K+, Tl+,
NH4+, and Cs+) and
ouabain protect strongly against thermal inactivation.
Ca2+, Ba2+, and La3+ ions do not
protect. Proteolysis experiments provide independent evidence that
disorganization can occur in stages, first in transmembrane segments
and then in extra-membrane segments of the fragments. Analysis of
selective dissociation of the M5/M6 fragment at 37 °C (Lutsenko, S.,
and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940), using a specific antibody, showed
that inactivation of Rb+ occlusion precedes dissociation of
the fragment, and only ~50% of the fragment is released when
occlusion is fully inactivated. In the presence of Ca2+
ions, occlusion is inactivated, but the M5/M6 fragment is not released.
The experiments demonstrate that occlusion is inactivated by disruption
of interactions between fragments of 19-kDa membranes, and only then
does the M5/M6 fragment dissociate. Interactions between the M5/M6 and
M7/M10 fragments seem to be essential for maintenance of
Rb+ occlusion.
 |
INTRODUCTION |
Recent studies of structure-function relations of the higher
P-type active cation pumps have focused on the characterization of
functional sites, for ATP and cations or inhibitors such as the cardiac
glycoside, or on experimental determination of transmembrane topology
(3). The consensus number of transmembrane segments in
-subunits of
Na,K-, H,K-, and Ca-ATPases is 10, and therefore, the focus of
attention is now shifting to the question of their arrangement in the
plane of the membrane. Site-directed mutagenesis or chimeric molecules
are being used intensively to characterize residues involved in cation
occlusion. Site-directed mutations suggest that carboxyl and other
oxygen-containing side chains of residues within transmembrane segments
M4-M6, and perhaps M8, ligate the occluded cations (4-6). Thus, the
transmembrane
-helices are arranged so as to create the cation
occlusion "cage."
A complementary biochemical approach for the study of cation sites and
the organization of transmembrane segments utilizes a preparation of
renal Na,K-ATPase extensively digested with trypsin (7, 8). This
preparation, referred to as "19-kDa membranes," consists of well
defined fragments of the
-subunit containing transmembrane segments
M7-M10 (apparent molecular mass of
19 kDa); the pairs M1/M2, M3/M4,
and M5/M6 (apparent molecular mass of 8-11.7 kDa); and the
-subunit partially split into a 16-kDa N-terminal and an
50-kDa C-terminal fragment. Cation occlusion and ouabain
binding are intact, but ATP binding is absent. These features
indicate that cation occlusion sites are located within transmembrane segments. Occluded cations (K+,
Na+, and congeners) (7, 8) and also ouabain (9) strongly protect the 19-kDa and smaller fragments of the
-subunit and also
the 16-kDa fragment of the
-subunit against trypsin. In the absence
of occluded cations or if Rb+ ions are displaced by
Ca2+ ions, Rb+ occlusion is destroyed, and all
the fragments are digested further to limit membrane-embedded peptides
(10). The segments digested away from 19-kDa membranes consist of tails
and loops between transmembrane segments outside the membrane. These
findings indicated that the fragments interact as a complex in which
several transmembrane segments cooperate to occlude the cations,
consistent with conclusions based on mutations. Occluded
Rb+ or Na+ or ouabain induces interactions
between fragments, stabilizing a compact structure that is inaccessible
to trypsin. Recently, we have demonstrated directly the existence of a
detergent-solubilized complex of all the fragments containing occluded
Rb+ ions and bound ouabain (11).
One objective of this work has been to investigate an observation that
incubation of 19-kDa membranes at 37 °C in the absence of occluded
cations leads to rapid loss of the ability to occlude Rb+
ions (1). Occluded Rb+ or Na+ ions protect
against this thermal inactivation as well as against further tryptic
digestion of the fragments. Indeed, trypsin digests only thermally
inactivated 19-kDa membranes, suggesting that the process involves
disorganization of the interacting fragments (10). A similar
implication has come from an observation that ouabain binding and
electrogenic Na+ binding are thermally inactivated at the
same rate (12). In recent experiments utilizing chymotrypsin to
demonstrate a specific role of the cytoplasmic tail of the
-subunit,
loss of Rb+ occlusion has also been found to be a result of
thermal inactivation (13). We have also proposed that loss of
Rb+ occlusion, following reduction of S-S bridges in the
-subunit (14), represents accelerated thermal inactivation of a
destabilized structure. Thermal inactivation of Rb+
occlusion appears to be a general phenomenon accompanying structural perturbations of 19-kDa membranes and can be used as a tool to investigate the structure of the cation-binding domain, the strength of
the interactions between different fragments, and the effect of ligands
or substrates on stability. Thermal inactivation of the native
Na,K-ATPase, with protection by K+ and Na+
ions, was described earlier (15, 16), but 19-kDa membranes, which lack
the cytoplasmic loops of the
-subunit, are much more thermolabile
than native enzyme and represent a simpler system to analyze the
factors determining the stability and interactions between
transmembrane segments.
Lutsenko and Kaplan (2) have shown recently that, upon incubation of
19-kDa membranes at 37 °C in the absence of Rb+ ions or
ouabain, the fragment containing the M5/M6 segments is selectively and
irreversibly released into the medium. Dissociation of the M5/M6
fragment is associated with partial inhibition of Rb+
occlusion, and loss of Rb+ occlusion has been described as
accompanying (2) or being caused by (17) dissociation of the M5/M6
fragment. Since occluded Rb+ ions prevent dissociation of
the M5/M6 fragment and also protect against thermal inactivation, it
has become necessary to characterize the relationship between the two
phenomena.
 |
EXPERIMENTAL PROCEDURES |
Na,K-ATPase was prepared from fresh pig kidney red outer medulla
by the rapid procedure described by Jørgensen (18). Protein was
determined by the method of Lowry et al. (19), and ATPase activity was determined as described previously (18). Specific activity
was in the range 13-20 units/mg of protein. Before use, the enzyme was
dialyzed at 4 °C against 1000 volumes of a solution containing 25 mM histidine, pH 7.0, and 1 mM EDTA (Tris).
Standard conditions for preparation of tryptic 19-kDa membranes were
described by Capasso et al. (8). After digestion, membranes
were washed, suspended, and stored in standard medium (25 mM imidazole, pH 7.5, and 1 mM EDTA), to which
2 mM RbCl was added.
Rb+ Occlusion Assay--
The Rb+
occlusion assays were performed as described by Shani et al.
(20). The medium contained, in a volume of 20-50 µl, 1 mM RbCl plus
5 × 106 cpm
86Rb+, 12.5 mM imidazole, pH 7.5, 0.5 mM EDTA, and 10-20 µg of 19-kDa membranes.
Thermal Inactivation of 19-kDa Membranes--
Membranes were
centrifuged and suspended in standard medium containing 0.1 mM RbCl and were then washed again and suspended in a
Rb+-free medium. The final free Rb+
concentration was estimated to be <1 µM. 19-kDa
membranes were incubated at 0.5-2 mg/ml, under the conditions
indicated in the figure legends, in a thermostatically controlled water
bath. At the indicated times, aliquots were placed on ice; reaction
medium containing 1 mM RbCl plus
5 × 106 cpm 86Rb+ was added; and
Rb+ occlusion was measured after a 60-min incubation at
0 °C or 5 min at 20 °C. Experimental points represent averages of
duplicate samples. Variability between duplicates was <10%.
Dissociation of the M5/M6 Fragment--
19-kDa membranes (150 µg/sample) were diluted with 1 ml of standard medium and centrifuged
at 250,000 × g for 1 h, and the pellet was
resuspended in ice-cold medium containing 10 mM Tris, 10 mM RbCl, or 1 mM CaCl2.
Phenylmethylsulfonyl fluoride (100 mM) was added to all
buffers to a final concentration of 1 mM. Samples were
incubated at 37 °C. Aliquots were removed at a certain time and
placed on ice. For electrophoretic analyses and immunoblots, the
samples were transferred to ice, and thermal inactivation was stopped
by addition of ice-cold standard medium containing 10 mM
RbCl. The samples were centrifuged at 250,000 × g for
1 h. Pellets were resuspended in standard medium containing 2 mM RbCl. Prior to SDS-polyacrylamide gel electrophoresis,
pellets were resuspended in standard medium and dissolved in 4% SDS,
and protein was precipitated by addition of 4 volumes of ice-cold methanol and stored overnight at
20 °C. The delipidated protein was collected by centrifugation for 30 min at 10,000 rpm in a Sorvall
centrifuge, dried under a stream of nitrogen, and dissolved in 10% SDS
or the sample buffer. The supernatants were collected, lyophilized, and
dissolved in the sample buffer. Equal amounts of delipidated membrane
protein (~100 µg for staining and 10 µg for immunoblotting) and
equivalent amounts of supernatant and pellet were applied per lane of
10% gels.
Gel
Electrophoresis--
Tricine1/SDS-polyacrylamide
gel electrophoresis was done essentially according to Schägger
and von Jagow (21) using either 1.5-mm-thick 10% gels (10% T and 3%
C separating gel (11.5 cm) plus 4% T stacking gel (1.5 cm)) or
1-mm-thick 16.5% gels (16.5% T and 6% C separating gel (20 cm), 10%
T spacing gel (2 cm), and 4% T stacking gel (1.5 cm)). Full details of
the electrophoresis procedure are given by Capasso et al.
(8). Scanning of transparencies of photographs of gels was performed
with a Molecular Dynamics 300A computing densitometer.
Immunoblots--
Immunoglobulins raised against the synthetic
peptide Leu815-Gln828 were supplied by Dr.
J. V. Møller (Aarhus University, Aarhus, Denmark), and
anti-Lys1012-Tyr1016, referred to as
anti-KETYY, was supplied by Dr. J. Kyte (University of California at
San Diego, La Jolla, CA). Rabbit antisera, prepared as described (22),
were raised against fragments of 19-kDa membranes (7, 8) and included
(i) anti-M1/M2, prepared from a 11.7-kDa fragment
(Asp68-Arg168) containing M1 and M2; (ii)
anti-
, prepared from a 16-kDa fragment (Ala5-Arg142) of the
-subunit; and (iii)
anti-
. Anti-peptide antibodies were also raised against the
synthetic peptides Leu337-Asn348 and
Ile263-Pro276, coupled to keyhole limpet
hemocyanin. Antibodies were diluted 1:100-400 in a solution of 1.5%
(w/v) bovine serum albumin in Tris-buffered saline solution. Samples
were delipidated, separated on 16.5 and 10% Tricine gels, and
electroblotted onto polyvinylidene difluoride paper according to
Matsudaira (23) using a Semi-Phor TE70 semidry transfer apparatus
(Hoefer Scientific Instruments). Immunoblot analysis was described
previously in detail by Capasso et al. (8).
Calculations--
Best fit parameters of theoretical equations
to experimental data were calculated by nonlinear regression analysis
using the Mathematica and MatLab software programs. First-order rate
constants for the thermal inactivation process were determined from
least-squares fits of the data to Equation 4 in the "Appendix." The
error estimates of the parameters are calculated by linear
approximation (24).
Calculation of Activation Parameters--
Activation parameters
were calculated as described by Huang (25). Activation energy
(Ea) values were estimated from slopes of Arrhenius
plots (ln k versus 1/T). Activation enthalpies (
H
) for each temperature were calculated
from the relation
H
= Ea
RT, where R (8.314 J·K
1·mol
1) is the universal gas
constant and T is the absolute temperature. Activation free
energies (
G
) were calculated according to
Equation 1,
|
(Eq. 1)
|
where kr (s
1) is the thermal
inactivation rate constant, h (6.6265 × 10
34 J·s) is the Planck constant, and
Kb (1.3805 × 1023
J·K
1) is the Boltzmann constant. Activation entropies
(
S
) were then calculated according to
Equation 2.
|
(Eq. 2)
|
Materials--
86RbCl was obtained from NEN Life
Science Products. Dowex 50W-X8 (100 mesh, H-form; converted to the Tris
form before use) was obtained either from Sigma or Fluka. Trypsin
inhibitor (type 1-S from soybean), bovine serum albumin (fraction V),
phenylmethylsulfonyl fluoride, thioglycolate, and molecular mass
markers (2.5-16.9 kDa) were from Sigma. Choline chloride
(recrystallized from hot ethanol) was obtained from Fluka. TPCK-treated
trypsin (bovine pancreas, 240 units/mg) was from Worthington. For
SDS-polyacrylamide gel electrophoresis, all reagents were of
electrophoresis grade and were from Bio-Rad. Polyvinylidene difluoride
paper was from Millipore Corp. Thallous acetate, lithium chloride, and
magnesium chloride were from BDH (Poole, United Kingdom). Cesium
chloride was from Fisher. Ammonium and potassium chlorides were from
Merck. Lanthanum chloride heptahydrate was from Aldrich.
 |
RESULTS |
Kinetics of Thermal Inactivation of Rb+
Occlusion--
Fig. 1 shows
representative time courses of thermal inactivation of Rb+
occlusion in the temperature range 25-45 °C and in the absence of
Rb+ ions. Because 19-kDa membranes are most stable when
stored in medium containing Rb+ ions, removal of
contaminating Rb+ ions by thorough washing of the membranes
(see "Experimental Procedures") was essential to ensure maximal
reproducibility of time courses for different preparations. If, for
example, the membranes were centrifuged only once and were then
resuspended in nominally Rb+-free solutions, the occlusion
was not fully inactivated at 37 °C, and the final level varied in
different experiments. Reports of incomplete thermal inhibition (2, 26)
of proteolyzed dog kidney enzyme might be explained by insufficient
washing of the membranes.

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Fig. 1.
Kinetics of thermal inactivation of
Rb+ occlusion in absence of Rb+ ions.
19-kDa membranes were incubated at the indicated temperatures in
standard medium in the absence of RbCl. At the indicated times, aliquots were withdrawn, and Rb+ occlusion was measured as
described under "Experimental Procedures."
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The time courses in Fig. 1 appeared to be biphasic, at least in the
range 25-35 °C, and the kinetics were therefore analyzed using the
two-step model of enzyme denaturation proposed by Lumry and Eyring (27)
(Equation 3),
|
(Eq. 3)
|
where N is the active native enzyme, U is the reversibly unfolded
inactive enzyme, and I is the irreversibly inactivated enzyme. The
model is described by the three rate constants
k1, k2, and
k3. Equation 4 in the "Appendix" assumes
that the remaining occlusion activity reflects the value of N and
describes a double exponential inactivation kinetic. The continuous
lines in Fig. 1 represent best fits to Equation 4. Best fit values of
k1, k2, and
k3 and also the amplitudes of the two phases
(A1 and A2) were calculated and collected together in Table
I. In the range 25-35 °C, the double
exponential kinetic provided a good fit to the data. At 40 and
45 °C, the kinetics could not be distinguished from a single
exponential process due to the shift of the relative amplitudes of the
two phases. Most curves at 37 °C were also fitted adequately by a
single exponential decay (data not shown).
Although values of k1,
k2, and k3 could be
obtained only over a limited range of temperatures and the fitting
errors are significant, we have estimated activation parameters
(
H
and
S
)
from the Arrhenius plots, for the values are informative with respect
to the mechanism of thermal inactivation (Fig.
2 and Table II). The positive values of
H
and
S
for
k1 and k3 reflect the
fact that k1 and k3 rose
as the temperature was raised. However, k2 did
not change appreciably between 25 and 35 °C (Table I), and thus,
H
is near to zero, whereas
S
has a negative value. The data are
consistent with the notion that k1 and
k3 represent disorganization processes but that
k2 represents a reorganization process (see
"Discussion").

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Fig. 2.
Arrhenius plots for thermal inactivation of
19-kDa membranes. Shown are Arrhenius plots of inactivation rate
constants (ln k) versus temperature
(1000/T, T in Kelvin). Plots are of the data in
Table I.
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Table II
Activation parameters for thermal inactivation of Rb+ occlusion
Activation parameters from the experimentally determined values of
k1, k2, and k3
were calculated from Equations 1 and 2 under "Experimental
Procedures."
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The Lumry-Eyring mechanism assumes the existence of a reversible
equilibrium between the N and U states, followed by an irreversible step to the I state. The obviously biphasic curve at 25 °C in Fig. 1
suggested that the U state might be significantly populated and that
inactivation might be reversible. As a test of this hypothesis, we
incubated 19-kDa membranes in the absence of Rb+ ions at
25 °C for 30 min and then transferred them to ice. Reaction medium
was added, and Rb+ occlusion was estimated over several
hours at 0 °C. As seen in Fig. 3, the
level of Rb+ occlusion, initially ~70% of the control
level, rose slowly and eventually reached ~95% of the control level.
By contrast, after inactivation of occlusion to 60% of the control
level by incubation at 37 °C for 1.5 min, no reversibility was
detected. The presence of Rb+ ions was not necessary for
reactivation of Rb+ occlusion at 0 °C (data not shown).
At 25 °C, it was also possible to observe reversibility of
inactivation after incubation with Rb+ ions for 1 h or
longer. The result at 25 °C is compatible with the Lumry-Eyring
model, but the model seems to break down at 37 °C and higher
temperatures (see "Discussion").

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Fig. 3.
Reactivation of Rb+ occlusion
after inactivation at 25 °C. 19-kDa membranes were suspended in
standard medium and incubated at 25 °C for 30 min or at 37 °C for
1.5 min. At the indicated times, aliquots were withdrawn and placed on
ice. The Rb+ occlusion of the samples was determined in the
presence of 5 mM RbCl plus 86Rb+ at
times from 0 to 24 h (see "Experimental Procedures").
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Protection against Thermal Inactivation by Occluded
Cations--
Previously, we reported that Rb+ or
Na+ ions protect against thermal inactivation, but aromatic
guanidinium and isothiouronium derivatives, which act as competitive
Na+ antagonists, do not protect (1, 28). In Fig.
4, a number of cations were tested for
protective effects at 37 °C. Rb+, K+,
Tl+, Cs+, NH4+,
and Na+ ions protected fully against thermal inactivation
under these conditions. The Km for Rb+
ions for protection was 89 µM (data not shown).
Li+ ions were ineffective under these conditions, although
in other experiments, Li+ ions at 50 mM were
able to protect against thermal inactivation at
30 °C.2 La3+
ions, which compete with Rb+ or Na+ ions (29),
and Ba2+ and Ca2+ ions, which also compete with
Rb+ ions in 19-kDa membranes (10), are ineffective.
La3+ and Ca2+ ions, like the aromatic
guanidinium and isothiouronium derivatives (1, 28), do not appear to be
occluded (10, 29). Thus, it appears that only cations that can be
occluded (K+, Na+, and congeners) can protect
against thermal inactivation.

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Fig. 4.
Selectivity of cations in protecting 19-kDa
membranes against thermal inactivation. 70 µg of 19-kDa
membranes were incubated at 37 °C for 5 min with 2 mM
RbCl (Rb), 10 mM KCl (K), 30 mM TlCH3COO (Tl), 50 mM
CsCl (Cs), 50 mM NH4Cl, 150 mM NaCl (Na), 50 mM LiCl
(Li), 100 mM LaCl3 (La),
2 mM BaCl2 (Ba), or 5 mM
CaCl2 (Ca). The control contained no addition
and was kept on ice. After incubation, 10 mM RbCl was
added, and the membranes were centrifuged and resuspended in standard
medium. 86Rb+ was then added, and occlusion was
measured. Protein concentrations of resuspended samples were
standardized by comparing A280.
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The ability of occluded Rb+ ions to stabilize against
thermal inactivation was analyzed in more detail in Fig.
5, which presents representative time
courses of loss of occlusion in the presence of a very high
concentration of Rb+ ions (50 mM). 50 mM RbCl was used because, in control experiments, the high
concentration was found to be necessary to saturate occlusion at
55 °C (data not shown). A striking feature is that occlusion was
significantly inactivated only at 45 °C and higher temperatures. Thus, Rb+ ions greatly slowed down thermal inactivation,
the rate at 45 °C being about the same as at 25 °C in the absence
of Rb+ ions (Fig. 1). The inactivation curves demonstrated
biphasic kinetics in the range 45-50 °C, and again the lines
represent best fits to Equation 4 in the "Appendix." At 52 °C
and higher temperatures, inactivation became monoexponential. Fitted
rate constants are presented in Table
III. Because values for
k1, k2, and
k3 were obtained only over a 5 °C temperature
span (45-50 °C) and the fitting errors are large, it was not
possible to obtain reliable quantitative values of activation
parameters (
H
and
S
) for comparison with those in the
absence of Rb+ ions. Nevertheless, comparison of the fitted
rate constants, with or without Rb+ ions, revealed
important qualitative features of the mechanism of action of the
Rb+ ions (see "Discussion").

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Fig. 5.
Time course of inactivation of
Rb+ occlusion in presence of Rb+ ions.
19-kDa membranes were incubated at the indicated temperatures in
standard medium containing 50 mM RbCl plus 10 × 106 cpm 86Rb+. At the indicated
times, samples were withdrawn and transferred to Dowex columns.
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Protection against Thermal Inactivation by Ouabain--
Ouabain
binds tightly to 19-kDa membranes in the presence of Mg2+
ions (K0.5 at 10 mM
MgCl2
1 µM) (12) and protects the 19-kDa fragment against tryptic digestion (9). Fig.
6 demonstrates that ouabain also protects
against thermal inactivation. Ouabain plus Mg2+ ions
inhibited Rb+ occlusion to
37% of the control level,
thus complicating the experiment. Nevertheless, in the sample incubated
at 37 °C for 5 min with ouabain plus Mg2+ ions, the
Rb+ occlusion was ~88% of that in the unincubated
sample. This value can be compared with the Rb+ occlusion
level of only 20% of control for the sample incubated at 37 °C
without ouabain plus Mg2+. Other control experiments showed
that ouabain alone (K0.5
1 mM)
slowed down the rate of Rb+ occlusion, but did not inhibit
the equilibrium level of Rb+ occlusion after incubation of
19-kDa membranes with reaction medium for 1 h at 20 °C (data
not shown). Therefore, protection experiments could also be done
conveniently in the absence of Mg2+ ions. Fig. 6 shows that
ouabain (5 mM) without added Mg2+ ions was
indeed able to protect strongly against thermal inactivation.

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Fig. 6.
Protection against thermal inactivation by
ouabain or ouabain plus Mg2+ ions. 70 µg of 19-kDa
membranes were resuspended in Rb+-free medium in the
absence or presence of 5 mM ouabain or 5 mM ouabain plus 10 mM MgCl2. Samples were
pre-equilibrated at 20-22 °C for 40 min in standard medium and
transferred to 37 °C for 5 min. Ice-cold reaction medium containing
5 mM RbCl plus 86Rb+ was added, and
occlusion was then measured after transfer of samples and incubation at
room temperature (20-22 °C) for 1 h.
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Fig. 7 (A and B)
presents a comparison of protection at 37 °C by Rb+ ions
(0.5 mM) and ouabain (5 mM), with both ligands
being present at close to saturating concentrations. The time courses
of thermal inactivation were similar in the presence of either 0.5 mM Rb+ ions or 5 mM ouabain and
were much slower than in the absence of either ligand. The combination
of 5 mM ouabain with 0.5 mM Rb+ was
even more effective. In Fig. 7B, we tested whether the
additional protection by ouabain could be observed even in the presence
of a very high concentration of Rb+ ions. As seen in Fig.
7B, this was found to be the case at the elevated
temperature of 47 °C.

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Fig. 7.
Comparison of effect of ouabain and
Rb+ on protection against thermal inactivation of
Rb+ occlusion in 19-kDa membranes. 19-kDa membranes
were incubated at 37 °C in standard medium containing 5 mM ouabain or 0.5 or 50 mM RbCl as indicated.
At the indicated times, aliquots were withdrawn, and Rb+
occlusion was measured as described for Fig. 6.
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Sensitivity of Fragments to Proteolysis Associated with Thermal
Inactivation--
Previously, we reported that, following irreversible
thermal inactivation of Rb+ occlusion at 37 °C, all
fragments of 19-kDa membranes can be digested further by trypsin or
chymotrypsin to limit membrane-embedded peptides (10, 13). The
experiment in Fig. 8 examined the
accessibility of the fragments to trypsin during a 30-min digestion at
25 °C, the condition for reversible inactivation (Fig. 3).
Rb+ occlusion was inactivated to 60% of the control level
in the sample without Rb+ and trypsin. The result was that
the fragments were insensitive to proteolysis and remained essentially
intact. Trypsin clipped three fragments known to contain the M1/M2
fragment (8) both in the absence and presence of Rb+ ions
(arrows). These clips are unrelated to the reversible
thermal inactivation since occlusion was not inactivated in the
presence of Rb+ ions at 25 °C. No cleavage of other
fragments was detected in the absence or presence of Rb+
ions. Thus, the extra-membrane tails or loops were essentially inaccessible to trypsin under conditions of reversible thermal inactivation.

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Fig. 8.
Insensitivity of 19-kDa membranes to tryptic
digestion at 25 °C. 19-kDa membranes were suspended in standard
medium with or without added Rb+ ions (10 mM)
and incubated at 25 °C for 30 min in the absence or presence of
TPCK-treated trypsin (1:10, w/w). The reaction was arrested with
trypsin inhibitor (5:1, w/w); the membranes were washed; and protein
was resolved on a 16.5% Tricine gel as described (13) (Fig. 1).
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Dissociation of the M5/M6 Fragment and Thermal Inactivation of
Rb+ Occlusion--
Figs. 9
and 10 present immunoblots of 19-kDa
membranes and supernatants, before and after treatments to thermally
inactivate Rb+ occlusion using
anti-Leu815-Gln828 near the C-terminal end of
the M5/M6 fragment, and, in addition, measurements of the
Rb+ occlusion levels. In 19-kDa membranes derived from pig
kidney, the fragment Gln737-Arg830 migrates on
Tricine gels with an apparent molecular weight of 8 kDa (8). In Fig.
9A, the membranes were suspended in a medium containing 10 mM RbCl or Tris-HCl and incubated at 37 °C for 10 min.
Incubation in the Tris-HCl medium was indeed associated with release of
the M5/M6 fragment into the medium. However, dissociation of the
fragment did not appear to be quantitative, with a significant proportion remaining in the membrane fraction. Rb+
occlusion was intact in the presence of RbCl, but was nearly fully
inactivated in the Tris-HCl medium. This lack of full dissociation of
the fragment and the nearly complete inhibition of Rb+
occlusion are significant discrepancies from the findings of the
previous work (2), in which quantitative dissociation of the fragment
and only ~50% inhibition of Rb+ occlusion were reported.
Therefore, we have examined the quantitative aspects in more detail.
Fig. 10 (A and B) presents immunoblots of the
membranes and supernatant after different times of incubation at
37 °C in the Tris-HCl medium and quantification of the amount of
M5/M6 fragment remaining in the membrane together with Rb+
occlusion levels. A preliminary experiment showed that the scanned signal from the M5/M6 fragment was proportional to the amount of
protein applied to the gel. Evidently, no more than ~50% of the
fragment was released at the longest incubation times, although Rb+ occlusion was completely inactivated. Furthermore,
inhibition of Rb+ occlusion preceded the release of the
fragment. Within 1 min of incubation at 37 °C, when Rb+
occlusion was ~30% inhibited, there was no detectable loss of the
fragment. Fig. 9B presents the results of a second type of experiment in which Rb+ occlusion was inhibited by
incubation with Ca2+ ions at 37 °C as described before
(10). Under this condition, we did not detect dissociation of the M5/M6
fragment, although Rb+ occlusion was fully inhibited. This
result constitutes a further difference from the results of Lutsenko
and Kaplan (2), who stated that Ca2+ ions were unable to
prevent dissociation of the M5/M6 fragment. In summary, the experiments
confirm dissociation of the M5/M6 fragment, but show that this
phenomenon is not a prerequisite for thermal inactivation of
Rb+ occlusion.

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Fig. 9.
Immunoblot analysis of dissociation of M5/M6
fragment at 37 °C in absence of Rb+ ions (A)
and in presence of Ca2+ ions (B). For
details of procedures and immunoblots using anti-Leu815-Gln828, see "Experimental
Procedures." The asterisks depict the positions of the
M5/M6 fragment. Lane M, molecular mass markers.
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Fig. 10.
Time course of dissociation of M5/M6
fragment and inactivation of Rb+ occlusion. For
details, see "Experimental Procedures" and the legend to Fig. 9.
A, immunoblot using
anti-Leu815-Gln828; B,
Rb+ occlusion and amount of M5/M6 fragment released into
the medium quantified by scanning the immunoblot.
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Experiments similar to those in Figs. 9 and 10 (data not shown), using
antibodies raised against the other fragments of 19-kDa membranes (see
"Experimental Procedures"), showed no significant dissociation of
the M1/M2, M3/M4, and M7/M10 fragments of the
-subunit; the 16-kDa
N-terminal fragment of the
-subunit; or the
-subunit. About
10-20% of the 50-kDa extracellular fragment of the
-subunit was
released into the medium. The time course was even slower than that for
release of the M5/M6 fragments, and it occurred also in the presence of
Ca2+ ions. Thus, there is no direct connection between
release of the two fragments. The lack of dissociation of the M3/M4
fragment is not in agreement with a recent claim that 60-70% of this
fragment dissociates together with the M5/M6 fragment (26).
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DISCUSSION |
Kinetic Analysis of Thermal Inactivation and Protection by Occluded
Cations and Ouabain--
The biphasic kinetics of thermal inactivation
of Rb+ occlusion (Fig. 1 and Table I, 25-35 °C) led us
to analyze the data by the two-step model for irreversible denaturation
(27), corresponding to stages in the disorganization of the fragments
(Fig. 11). For the temperature range
25-35 °C and in the absence of Rb+ ions, the good fit
of the biphasic curves in Fig. 1 to Equation 4 in the "Appendix"
provides one line of evidence consistent with this mechanism. A second
line of evidence compatible with the model is the reversible
inactivation observed at 25 °C (Fig. 3). By applying the fitted rate
constants at 25 °C (Table I) to Equations 4 and 5 in the
"Appendix," the estimated fractions of N, U, and I after 30 min are
75, 17, and 8%, respectively. The predicted initial and final levels
of occlusion in the reversibility experiment are 75 and 93% of control
levels, respectively. These values are not inconsistent with the data
in Fig. 3. Large positive values of both
H
and
S
for k1 and
k3 are typical of entropy-driven disorganization
processes, whereas a value of
H
close to
zero and a strongly negative
S
provide a
strong indication that k2 reflects a
reorganization process (Table II). That the N
U equilibrium
involves disorganization and reorganization can also be inferred from
estimates of
H0 and
S0 based on the calculated equilibrium
constant (k1/k2) using
the values of k1 and k2
in Table I (ratios are 0.25, 1.55, and 3.16 at 25, 30, and 35 °C,
respectively).
H0 and
S0 derived from a van't Hoff plot are 46 kcal·mol
1 and 153 cal·K
1·mol
1, respectively. The two-step
mechanism seems to relax into a one-step inactivation mechanism (N
I) at ~37 °C and higher temperatures since the kinetics were
fitted adequately by a single exponential decay. Conversion of the
two-step to a one-step mechanism implies that the U form is present
only in negligible amounts. Lack of reversibility of inactivation at
37 °C (Fig. 3) is consistent with absence of the U form.
Disappearance of the U form as a significant intermediate could occur
if k3 rose and k2 fell
sharply over a narrow temperature range, implying that the
disorganization process is highly cooperative.

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Fig. 11.
Schematic model with stages of thermal
inactivation of Rb+ occlusion. The model depicts the
stages of thermal disorganization of a four-helix bundle. N,
gates are closed, and two ions are occluded. Interactions between
extra-membrane tails are intact. U, gates are open, and
occluded cations dissociate. Interactions between extra-membrane tails
are intact. I, the complex is fully disorganized. After
thermal inactivation, the M5/M6 fragment dissociates.
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To explain biphasic kinetics, more complex schemes, invoking the
existence of two populations of enzyme molecules or two pools of
occluded Rb+ ions within the same molecule (26), might be
considered. Such mechanisms would have to assume both different rates
of thermal inactivation and interconversion between the pools since the
amplitudes of the phases are strongly affected by temperature (Table
I). Apart from their additional complexity, neither of these mechanisms predicts reversible inactivation (Fig. 3), and they are therefore unlikely explanations of the results described here.
Occluded cations protect strongly against thermal inactivation, as
shown in Figs. 4 and 5. In the presence of Rb+ ions, the
two-step Lumry-Eyring mechanism fits the data between 45 and 50 °C,
but at 52 °C and higher temperatures, it appears to break down, and
a single exponential decay suffices. It is of interest to compare
fitted rate constants (k1,
k2, and k3) without and
with Rb+ ions (Tables I and III), although the comparisons
are qualitative or only semiquantitative due to the limits on accuracy
of the data and lack of overlap of the temperature ranges. A direct
experimental comparison was possible for k1 at
45 °C (1.02 ± 0.23 min
1 without Rb+
ions versus 0.22 ± 0.11 min
1 with
Rb+ ions). For k1 at other
temperatures and k2 and
k3, the comparison is indirect due to the lack
of overlap of accessible temperatures (25-35 °C without
Rb+ ions and 45-50 °C with Rb+ ions).
Inspection of the values of k1,
k2, and k3 at 45 °C
with Rb+ ions (Table III) and at 35 °C without
Rb+ ions (Table I), together with the observed tendency of
k1 and k3 to rise and of
k2 to fall as temperature rises, makes it clear that Rb+ ions must lower k1 and
k3 and raise k2. An
effect of Rb+ ions to suppress k1 is
not surprising. However, an effect of Rb+ ions on
k3 and k2 is paradoxical
because the implication is that Rb+ ions bind to the U
form, whereas the two-step model assumes that only the N form is able
to occlude Rb+ ions. To resolve this paradox, we propose
that the product of the first step of thermal inactivation, U, is only
partially disorganized and that Rb+ ions are still able to
bind with a low affinity. Due to the rapid dissociation of the
Rb+ ions, occlusion of Rb+ is not detectable,
but the low affinity binding of the Rb+ ions elevates
k2 and suppresses k3. The
second step (U
I) leads to a more disorganized conformation with no
Rb+ recognition. These concepts are not incompatible with
the schematic structural model in Fig. 11.
Protection against thermal inactivation by ouabain suggests that
ouabain and Rb+ ions induce similar stabilizing
interactions. The combination of ouabain and Rb+ produces a
further stabilization (Fig. 7). Ouabain reduces the rate of
dissociation of occluded Rb+ ions (11, 30), and a part of
the additional stabilization could be due to this effect, but the
finding that ouabain confers extra stabilization even at very high
concentration of Rb+ ions (Fig. 7) shows that it must
induce additional interactions. The latter conclusion might explain our
finding that a combination of Rb+ ions and ouabain was
required to stabilize a detergent-solubilized complex of fragments of
19-kDa membranes (11).
Model for Thermal Inactivation of Occlusion and Dissociation of the
M5/M6 Fragment--
The kinetics of thermal inactivation (Figs. 1 and
3 and Table I-III) and proteolysis experiments (Fig. 8) provide
complementary evidence for two stages of disorganization of the
occlusion cage. These stages and the subsequent dissociation of the
M5/M6 fragment are depicted in the schematic model of transmembrane
helices in Fig. 11. The model also indicates that, at 37 °C and
higher temperatures without Rb+ ions (or at 52 °C or
higher temperatures with Rb+ ions), the intermediate U form
is not significant, and a one-step mechanism of thermal inactivation
suffices. The reversible step (N
U) appears to involve
disorganization within the membrane domain since the extra-membrane
loops and tails are inaccessible to proteases under conditions of
reversible inactivation (Fig. 8), suggesting that the secondary
structure and interactions of these segments are largely intact. An
important implication is that barriers or "gates" to dissociation
of occluded cations are located within transmembrane segments and are
broken in this first step. The U form might be thought of as a
"molten globule," in which the tertiary structure is disrupted, but
the overall folding and secondary structure are largely native (31).
The subsequent irreversible step (U
I) involves more extensive
disorganization in which the interactions between extra-membrane tails
and loops and their secondary structure are disrupted, and so these
segments become accessible to proteases, as we have described
previously (10, 13). The strong protection of occluded Rb+
and other cations against the disorganization depicted in Fig. 11
implies that the stabilizing effects are not restricted to the occlusion cage, but occur both within the membrane domain and outside
the membrane, i.e. these are global effects. The latter conclusion is consistent with results of proteolysis experiments using
19-kDa membranes that show that occluded cations induce substantial
structural changes in fragments of both
- and
-subunits (9,
10).
Thermal inactivation of native Na,K-ATPase, which is strongly protected
by K+ ions (15, 16), may also proceed by disorganization of
transmembrane segments. Indeed, in experiments using intact renal
microsomes, we have demonstrated directly a change in transmembrane
topology in the C-terminal region of the
-subunit accompanying
thermal inactivation of Na,K-ATPase activity or Rb+
occlusion (32). Rb+ ions protected strongly against both
inactivation of function and disorganization of transmembrane segments
(32).
The experiments in Figs. 9 and 10 confirm the observation of
selective dissociation of the M5/M6 fragment, consistent with a central
role of transmembrane segments M5 and M6 in cation occlusion (2, 33).
However, differences in the experimental findings from those reported
by Lutsenko and Kaplan (2) affect the interpretation of this
phenomenon. In our experiments, only ~50% of the M5/M6 fragment was
released when occlusion was fully inactivated, and inactivation of
occlusion preceded release of the fragment. In addition, no release of
the fragment accompanied thermal inactivation in the presence of
Ca2+ ions. A possible explanation of these differences is
that detection is more sensitive with use of the antibody compared with
N-terminal sequencing of the fragment as used by Lutsenko and Kaplan
(2). At 25 °C, the condition for reversible inactivation, the M5/M6 fragment was not released (data not shown), demonstrating also that
dissociation is not necessary for thermal inactivation.
The central conclusion from the results in Figs. 9 and 10 is that
dissociation of the M5/M6 fragment is not the direct cause of loss of
Rb+ occlusion, but rather thermal inactivation disrupts
protein-protein interactions, inactivating the Rb+
occlusion, and then the M5/M6 fragment is released into the medium (Fig. 11). A corollary is that dissociation of the M5/M6 fragment cannot be taken as evidence for a "piston-like" movement in the normal cation transport cycle (2, 33) because the fragment is
dissociating from an inactive disorganized form of the enzyme. A
finding that the M5/M6 fragment of H,K-ATPase is released from the
membrane after washing with carbonate at pH 10 (34) is also suggestive
of an alkali-dependent disorganization of the fragments prior to dissociation.
Interactions between the M5/M6 and M7/M10
Fragments--
Experiments using 19-kDa membranes have shown that both
occluded cations (K+, Na+, and congeners) and
ouabain protect against thermal inactivation of Rb+
occlusion (described here and see also Refs. 1 and 10); they protect
the M7/M10 fragment against digestion by proteases (7-9); and they
both also protect against dissociation of the M5/M6 fragment (2). These
findings provide suggestive, if indirect, evidence that both
Rb+ ions and ouabain induce stabilizing interactions
between the M5/M6 and M7/M10 fragments and that thermal inactivation
results from disruption of these interactions. Further indirect
evidence for interactions between the M5/M6 and M7/M10 fragments comes from observations that dissociation of the M5/M6 fragment results in
exposure of cysteine residues in the M7/M10 fragment to selective chemical modification (2, 17).
We have recently obtained direct evidence for interactions between the
M5/M6 and M7/M10 fragments of 19-kDa membranes based on covalent
cross-linking.3 This work
provides a stronger indication that interactions between the M5/M6 and
M7/M10 fragments are essential for maintaining intact the cation
occlusion and transport structures.
We thank Prof. J. V. Møller for
providing anti-Leu815-Gln828.