(Received for publication, December 26, 1995; and in revised form, February 14, 1996)
From the
The purified plasma membrane Ca-ATPase is
fully activated through the enzyme concentration-dependent
self-association at physiologically relevant Ca
concentrations (Kosk-Kosicka, D., and Bzdega, T.(1988) J.
Biol. Chem. 263, 18184-18189; Kosk-Kosicka, D., Bzdega, T.,
and Wawrzynow, A.(1989) J. Biol. Chem. 264,
19495-19499). We have previously shown that the
Ca
-ATPase activity of the oligomeric enzyme is
independent of calmodulin, in contrast to another active enzyme
species, a presumable monomer, that is activated by calmodulin binding.
Presently, we have succeeded in determining the molecular mass of the
two active enzyme species by equilibrium ultracentrifugation. For the
calmodulin-dependent species, the molecular mass is 170 ± 30
kDa, which is consistent with predominantly monomeric
Ca
-ATPase with bound calmodulin. The molecular mass
of calmodulin-independent oligomers is 260 ± 34 kDa, indicating
that they are dimers. Results of experiments performed under different
calcium and potassium concentrations and in the presence of dextran
that causes molecular crowding verify a strict Ca
requirement of the dimerization process. We conclude that the
active species of the Ca
-ATPase are a
monomer-calmodulin complex and a dimer.
The erythrocyte Ca-ATPase is a plasma membrane
Ca
pump responsible for Ca
removal
from the cytoplasm to maintain the steep Ca
concentration gradient. The protein exhibits an M
of 134,000 upon SDS-gel electrophoresis, in agreement with its
primary sequence (for review, see (3) ). It has been shown
recently that the erythrocyte enzyme consists of two isoforms encoded
by two different genes designated PMCA1 and
PMCA4(4, 5, 6) . The isoforms are expressed
in all of the tissues recently tested by Western blot analysis and are
believed to be housekeeping pump isoforms, whereas PMCA2 and PMCA3,
which are absent from erythrocyte plasma membrane, are more specialized (7) .
We have demonstrated that the Ca pump isolated from human erythrocytes undergoes reversible,
enzyme concentration-dependent oligomerization that reaches maximal
levels at 30-40 nM enzyme(1, 2) . The
oligomerization process produces a highly cooperative
Ca
-regulated activation of the enzyme at
physiologically relevant calcium concentrations (K
50
nM)(2, 8) . Using measurements of
fluorescence resonance energy transfer between appropriately labeled
enzyme molecules, we were able to differentiate two active species of
the enzyme, which we defined as oligomers and monomers with bound
calmodulin(2) . Calmodulin can activate the enzyme, apparently
by binding to the monomeric form. Calmodulin does not increase the
activity of enzyme oligomers, and the resonance energy transfer
measurements indicate that calmodulin does not dissociate preformed
oligomers(9) . Instead, calmodulin-monomer complex formation
appears to be competitive with a monomer to oligomer transition, and
both enzyme species are fully active. The extent of oligomerization and
thus the structure of the active forms of the enzyme could not be
determined by the methods used previously, and it is this point that is
addressed here.
Some of these data have been reported in a preliminary form(10) .
Egg yolk phosphatidylcholine (P5763) and CNBr-activated
Sepharose 4B and dextran (D4751) were purchased from Sigma, and
octaethylene glycol dodecyl ether (CE
) was
obtained from Nikko (Tokyo, Japan). Coupling of bovine calmodulin to
Sepharose was performed in accordance with Pharmacia Biotech Inc.
instructions as described earlier(1) .
The methods used for
preparation of erythrocyte ghost membranes, purification of the
Ca-ATPase from the membranes, and determination of
protein and Ca
concentrations were as described
previously(1, 11) . Free Ca
concentrations were calculated from total calcium and EGTA
concentrations based on the constants given by Schwartzenbach et
al.(12, 13) . Total calcium was measured by
atomic absorption.
There were two technical problems posed in
the application of this technique to the Ca-ATPase
system. First, the low optical density presented by 50 nM enzyme meant that detection of signal over background was
difficult. Second, the lability of the enzyme upon dilution in standard
buffer conditions placed limits on the possible length of experiments
and therefore the available time to reach equilibrium.
The lability of the enzyme required that analysis be performed as quickly as possible after thawing and diluting the enzyme. This requirement was accommodated by using small sample volumes (40 µl) that yielded short column heights (<1 mm) and consequently rapid attainment of equilibrium(15) . These small samples could be accommodated in both instruments used. About 4 h of centrifugation proved to be enough to reach equilibrium. Under these conditions, the enzyme lost about 30% activity during this time, as shown in Table 1.
The problem presented by low optical density was reduced by averaging multiple scans. In the case of the model E data collected at 280 nm, multiple scans of each cell were taken at equilibrium, aligned at the meniscus, and points at corresponding radial positions were averaged. In the case of the XLA data collected at 230 nm, data at each radial position were averages of several determinations, as set by the control parameters of the scans. Multiple experiments were performed in this way on both instruments.
where r is the radius of a reference point
such as the base of the cell and c
is the
concentration of the molecule at that point. M is the weight
average molecular mass of the molecule, B is the buoyancy
factor (discussed below), and C =
/2RT, where
is the angular velocity, R is the gas constant, and T is the absolute
temperature. The data (absorbance versus radius) will then be
of the form
where A is the absorbance at radius r, A
is the absorbance at the base of
the cell due to the protein, and e is a base-line absorbance
term. The buoyancy factor B is defined as
where v is the partial specific volume of the molecule
and is the solution density. If the protein has bound detergent,
an additional term must be added to correct for the buoyancy of the
detergent, and B becomes
where is the amount (g) of detergent bound per
g of protein and v
is the partial specific volume
of the detergent.
In the absence of measured values for the amount
of bound detergent, , centrifugation is commonly
carried out in solutions of varying density, allowing extrapolation to
the condition where the solution density
=
1/v
. At this point, the second term in B becomes 0, and only the protein partial specific volume, v
, influences the observed buoyant molecular
weight(16) . The same result is obtained if the detergent used
has partial specific volume equal to 1/
. This is the case here
since the buffer is not much different from water (measuring
= 1.014 g/cm
) and the detergent,
C
E
, has partial specific volume, v = 0.973(17) . With this detergent in low salt
buffer, the bound detergent makes little contribution to the buoyancy
factor B, and thus the second term in B may be taken
as 0(18) . Data were thus fit to with B defined as in .
Data from the model E were analyzed using the non-linear least squares fitting routines of MLAB (Civilized Software, Bethesda, MD) and a logarithmic form of , as described previously(15) . Error estimates given are the standard error estimates given by the MLAB fitting routine. Data from the XLA were analyzed using the software provided by Beckman. Model E data were also analyzed using the Beckman software with the same fitting results as obtained with MLAB. Standard error estimates are not calculated by the Beckman fitting routines and were estimated in a different way. Following data collection, the rotor was run at higher speed for approximately 40 min in order to record the base-line (non-sedimentable) absorbance. The mean base-line absorbance and its associated standard deviation were calculated. The data were then fit three times with the base-line value held fixed at the mean value, at the mean +1 S.D., or at the mean -1 S.D. The best fit values obtained were taken as estimates of molecular mass ± S.D. The partial specific volume of the protein was taken as v = 0.73. In experiments in which a complex with calmodulin was examined, the complex was also assumed to have v = 0.73 since the v calculated (19) for bovine calmodulin was 0.72.
We undertook the determination of the molecular mass of
different forms of the erythrocyte Ca-ATPase, the
oligomeric form with calmodulin-independent activity and the form with
calmodulin-dependent activity previously termed monomer. To
characterize the enzyme, we have applied analytical ultracentrifugation
that is a rigorous means of determining the mass of covalent and
non-covalent macromolecular complexes(20, 21) . Due to
the technical difficulty of obtaining data from samples containing very
diluted proteins, the lowest enzyme concentration at which we could
determine the molecular mass of the Ca
-ATPase was 50
nM. At this concentration, the enzyme is fully oligomerized as
indicated by Ca
-ATPase activity and fluorescence
resonance energy transfer measurements(1, 2) . To
circumvent the very low signal to noise problem of direct measurements
at the low protein concentrations typical of monomers (K
= 8-15 nM enzyme), we
performed experiments under several conditions listed in Table 2in which calcium and potassium concentrations have been
changed, calmodulin was added, and molecular crowding was achieved by
the addition of dextran.
The first question we approached was the
oligomerization state of the enzyme under conditions that yield full
Ca-ATPase activity in the absence of calmodulin (set
1 in Table 2). This was addressed by determining the molecular
mass of the 50 nM enzyme in the standard buffer under
conditions at which the enzyme concentration-dependent activation of
the Ca
-ATPase by self-association has been
demonstrated(1, 2) . Typical gradients recorded in the
model E at 280 nm and in the XLA at 230 nm are presented in Fig. 1. The best fit masses for these data sets were averaged
and are presented in Table 3(set 1). The data from the model E
(271 ± 28 kDa) and the XLA (252 ± 42 kDa) are both
consistent with a predominantly dimeric form of the enzyme whose
calculated molecular mass is
270 kDa.
Figure 1:
The enzyme is dimeric at concentrations
that yield full activity. Enzyme solutions were prepared at 50 nM in standard buffer (Tris maleate, pH 7.4, 120 mM KCl, 17
µM free Ca, 1 mM EGTA, 8 mM MgCl
, 150 µM C
E
), i.e. standard reaction
mixture (condition 1 in Table 2and Table 3), and
centrifuged to equilibrium in the model E (A) or the XLA
ultracentrifuge (B). Attainment of equilibrium was determined
by difference scans as described under ``Materials and
Methods.'' At equilibrium, scans were taken at 280 nm (A)
or 230 nm (B). The scans presented are typical of those
obtained from multiple runs. The upper section of each panel
presents the data and fit and the lower sections show the
differences between the data and the fit. In panel A, the solid curve shows the calculated gradient of absorbance for
the best fit mass of 255 kDa. In panel B, the solid curve shows the calculated gradient corresponding to the best fit mass
of 288 kDa. O.D., optical density.
The second condition (Table 2, set 2) addresses the effect of calmodulin. In the presence of calmodulin, the ATPase is fully active, but fluorescence energy transfer measurements between labeled enzyme molecules indicate that the enzyme does not oligomerize under these conditions(9) . In agreement with the fluorescence measurements, 50 nM enzyme, when added to superstoichiometric (100 nM) calmodulin, no longer behaves in the centrifuge as a dimer (Table 3, set 2 and Fig. 2). The apparent molecular mass is lowered as compared with the results obtained with 50 nM enzyme alone. Results with both instruments yield a mass of about 170 kDa, which is consistent with a complex between the enzyme monomer and calmodulin (predicted mass of 151 kDa).
Figure 2:
Calmodulin forms a complex with monomeric
Ca-ATPase. Enzyme solutions were prepared exactly as
in Fig. 1except that calmodulin was added at a concentration
(100 nM) superstoichiometric to that of the enzyme (50
nM), i.e. condition 2, Table 2. Panel A presents a typical scan taken at 280 nm with the model E, and the solid curve shows the calculated gradient for the best fit
mass of 190 kDa. Panel B presents a typical scan taken at 230
nm with the XLA. The solid curve here shows the gradient for
the best fit mass of 208 kDa. In both panels, the lower sections show the residuals from the fits. O.D., optical
density.
In the third condition
tested (Table 2, set 3), calcium and potassium concentrations
were decreased compared with standard conditions (Table 2, set
1). At low K/Ca
concentrations, the
activity of the 50 nM enzyme is very low and is stimulated by
calmodulin. This behavior is typical of the so-called monomeric enzyme (1, 9) (compare also with set 4 in Table 2). The
molecular mass of about 180 kDa (Table 3, set 3) is lower than
that calculated for dimers. The finding indicates that the observed low
activity and its dependence on calmodulin are due to the fact that
monomers are a dominant part of the enzyme population under these
conditions.
More information on enzyme association at the low
K/Ca
concentrations condition was
derived from experiments with dextran. Dextran was used because its
addition to the Ca
-ATPase at 15 nM concentration (at which concentration the
Ca
-ATPase requires calmodulin for activation,
suggesting that it is monomeric) induced self-association of the enzyme
as judged by fluorescence resonance energy transfer
experiments(22) , and thus led to full, calmodulin-independent
activity (Table 2, set 4). Dextran had no effect on the 50 nM (dimeric) enzyme (Table 2, set 1; for details, see also (22) ). Addition of dextran to the 50 nM enzyme at low
K
/Ca
concentrations in the absence
of calmodulin (Table 2, set 3) did not increase
Ca
-ATPase activity, indicating that the apparent
molecular mass of about 180 kDa (Table 3, set 3) is that of
monomers that are prevalent under these conditions. Apparently at low
calcium, the enzyme simply cannot self-associate. Also, at low enzyme
concentration (15 nM) in the presence of low
K
/Ca
, the
Ca
-ATPase activity (Table 2, set 5) is not
fully activated in the presence of dextran, again indicating that
insufficient calcium hinders dimerization. Potassium concentration
appeared to have no significant effect on enzyme dimerization since the
apparent molecular mass of the 50 nM enzyme in the presence of
high K
(but also in the absence of
Ca
) was again about 170 kDa (Table 3).
The application of analytical centrifugation with the
modifications introduced previously to analyze the dimerization of the
labile protein tubulin (14, 15) made it possible to
finally determine the sizes of the two species of the plasma membrane
Ca-ATPase whose contribution to enzyme activity was
demonstrated several years
ago(1, 2, 8, 9, 23) . We
have now established that the fully active calmodulin-independent
enzyme is dimeric. We have also confirmed that the species activated by
calmodulin is indeed a monomer-calmodulin complex.
Previous attempts
to establish the sizes were hampered by the low enzyme concentrations
at which the transformation from calmodulin-dependent monomers to the
calmodulin-independent dimers of Ca-ATPase occur (K
= 8-15 nM enzyme, see (1) and (2) ).
The fact that the centrifuge data
were collected on two different instruments, which was a necessity due
to a coincidence in timing, proved to be an advantage allowing us to
compare two independently obtained data sets. The model E and the model
XLA are quite different instruments, but they yielded comparable
results. The model E has a very wide spectral bandpass and was
essentially limited to 280 nm for these experiments. This presented a
problem of low absorbance at this wavelength of the
Ca-ATPase at 50 nM. The XLA, in contrast,
has a narrow spectral bandpass and allowed wavelengths as low as 190 nm
to be used for analysis. Additionally, the XLA data acquisition system
is considerably simpler to use and much more powerful than the one we
had on the model E. Lower wavelengths presented a significantly higher
sample absorbance but also presented a significantly increased buffer
absorbance background. A compromise of 230 nm was chosen. This allowed
a reasonable sample absorbance to be recorded but introduced
significant noise in the base-line determination, requiring a different
approach to error estimation. Despite these differences and the
problems common to both, experiments with the two instruments provided
results that indicate the same biological result. The molecular mass of
the enzyme in the absence of calmodulin is consistent with the
predominance of dimers (Fig. 1), while the molecular mass of the
enzyme in the presence of calmodulin is consistent with
monomer-calmodulin complexes (Fig. 2). Within the 4 h required
for equilibration, the molecular mass was never significantly higher
than that expected for enzyme dimers, thus indicating absence of a
meaningful amount of oligomers of a higher size than of dimers.
In
addition, the analytical centrifugation confirmed two previous findings
that have been made using fluorescence spectroscopy methods
(fluorescence energy transfer and polarization measurements). First,
activation by enzyme dimerization is a Ca-dependent
process(2) . Second, our data indicated that the equilibrium
between enzyme monomers and dimers and the availability of calmodulin
to the two enzyme forms determine the activation pathway of the
purified Ca
-ATPase(9) . Accordingly, the
distinctly lowered molecular mass of 50 nM enzyme that was
centrifuged to equilibrium at insufficient Ca
indicates a predominantly monomeric population in agreement with
the extent of dimerization that is limited by the availability of
Ca
. Second, in the presence of superstoichiometric
calmodulin at optimal Ca
, the enzyme at 50 nM concentration after 4 h equilibrated to yield molecular mass that
is consistent with monomer-calmodulin species rather than with dimers (Fig. 2). This finding could be plausibly explained by the
differences in the affinities of the enzyme-calmodulin and the
enzyme-enzyme interactions. The apparent affinity of enzyme monomer for
calmodulin is higher (K
= 1.6-3.5
nM) (1, 24) than its affinity for another
monomer (K
= 8-15 nM enzyme)(2) . While the affinity for calmodulin binding to
the dimer is not known, there are multiple experimental demonstrations
of such binding(1, 23) . In fluorescence spectroscopy
measurements of 5-min duration, an addition of calmodulin to the dimers
formed between donor-labeled and acceptor-labeled enzyme molecules did
not decrease energy transfer, suggesting that calmodulin did not
dissociate them. However, calmodulin prevented increased energy
transfer resulting from dimerization if it was added to 28 nM donor-labeled enzyme before the addition of 28 nM acceptor-labeled enzyme(9) .
The results generated by
analytical ultracentrifugation are consistent not only with
fluorescence spectroscopy measurements but also with the results
produced by another approach, induction of enzyme self-association by
molecular crowding in the presence of dextran. We have previously
demonstrated that dextran induces oligomerization of enzyme molecules
at concentrations that are too low (beginning with 6 nM enzyme) for significant self-association under standard
conditions(22) . At present, we have shown that at insufficient
Ca even dextran cannot force the enzyme to dimerize.
In conclusion, the two active species of erythrocyte
Ca-ATPase identified 7 years ago can be now
recognized as a dimer and a monomer-calmodulin complex.