(Received for publication, October 2, 1995; and in revised form, November 21, 1995)
From the
The P-type Ca-ATPase from Flavobacterium
odoratum has been purified to homogeneity and characterized.
Inside-out membrane vesicles were extracted with
C
E
, followed by ammonium sulfate
fractionation, centrifugation through two successive 32-48%
glycerol gradients, and DE52 ion exchange chromatography. The purified
Ca
-ATPase consists of a single polypeptide. It
migrates electrophoretically with an apparent molecular mass of 60,000
Da, consistent with the phosphorylation pattern originally reported in
membrane vesicles. This single polypeptide is functional and capable of
calcium-dependent vanadate-sensitive ATP hydrolysis and of forward and
reverse phosphorylation. Maximum hydrolysis activity occurs at pH 8.0,
with a specific activity of
75 µmol of ATP hydrolyzed
min
mg
protein. The purified
Ca
-ATPase has an apparent K
for calcium of 1.5 µM and for ATP of 90
µM. Vanadate strongly inhibits the activity with an
IC
of 0.6 µM. The prokaryotic
Ca
-ATPase is insensitive to the SR
Ca
-ATPase inhibitors fluorescein isothiocyanate,
thapsigargin, and cyclopiazonic acid. It is rapidly phosphorylated by
[
-
P]ATP in a calcium-dependent
vanadate-inhibited manner and can be phosphorylated by P
in
both the presence and absence of calcium.
In eukaryotic cells, the utilization of calcium in signal
transduction requires tight regulation of free calcium concentration.
Maintenance of low calcium is achieved by both ATPases and antiporters.
The Ca-ATPase of sarcoplasmic reticulum has been
studied in the greatest detail and becomes the model for the analysis
of structure-function relationships in ion transport. However, for the
vast majority of bacterial species examined, Ca
transport is carried out solely via secondary transport, by
Ca
/H
or
Ca
/Na
antiporters(1, 2) . To date, ATP-driven
vanadate-sensitive calcium accumulation has been identified only in (a) three species of Gram-positive bacteria, Enterococcus(3, 4, 5) , (b)
the Cyanobacterial species Anabaena variabilis and Synechococcus sp.(6, 7) , and (c)
the Gram-negative bacteria Flavobacterium
odoratum(8, 9) . In the former cases, either low
activity or failure to stably extract from the membrane has precluded
purification and further study of the ATPases. However, the abundance
of stable detergent-extractable Ca
-ATPase activity in F. odoratum has allowed it to be the most completely
characterized of the prokaryotic
Ca
-ATPases(10) . Its K
for Ca
and ATP and IC
for
vanadate are very similar to that of the sarcoplasmic reticulum
Ca
-ATPase(8) . The enzyme is rapidly
phosphorylated by [
-
P]ATP in a
calcium-dependent manner. Both the forward and reverse reaction cycle
intermediates have been examined. Although the prokaryotic pump is
functionally similar to its eukaryotic counterpart, the forward limb of
its reaction cycle is mechanistically distinct. Unlike the eukaryotic
P-type ATPases, the forward reaction of the prokaryotic
Ca
-ATPase appears to be ordered, and the high
affinity Ca
binding site is not accessible until ATP
binds(9, 10) .
Clearly, comparison of the molecular
mechanism of the well characterized eukaryotic P-type ATPases should
provide extremely valuable information concerning both evolution and
function. Only two prokaryotic P-type ATPases have been purified to
homogeneity. The best characterized is the high affinity potassium pump
(Kdp-ATPase) of Escherichia coli(11) . The Kdp-ATPase
consists of three subunits of 60,000, 72,000, and 20,000
Da(12, 13) . Preliminary evidence suggests that the
functional complex is oligomeric with respect to the subunits
(AB
C
), however it is subunit B that
bears homology to the eukaryotic ATPase-conserved sequences and that
demonstrates a phosphorylated intermediate. The second purified pump is
a K
-ATPase that has been isolated from the
Gram-negative bacterium Enterococcus hirae as a single
polypeptide with an M
of 78,000(14) .
In this report, we describe the purification of the F. odoratum Ca-ATPase to homogeneity. By monitoring
calcium-dependent ATP turnover and the formation of an alkaline-labile
phosphointermediate, a purification factor of 1800 was achieved, using
glycerol gradient centrifugations and column chromatography. The ATPase
is a single polypeptide with an apparent M
of
60,000. The isolated enzyme is very stable and capable of forming an
alkaline-labile phosphointermediate in the presence of either ATP or
inorganic P
. It demonstrates kinetics of hydrolysis similar
to the SR Ca
-ATPase and is the smallest P-type ATPase
as described to date.
The
apparent K for Ca
and ATP and
the IC
for vanadate were determined for the purified
Ca
-ATPase. For the determination of the K
for calcium, the free calcium concentration in
the reaction buffer was adjusted using calcium-EGTA mixtures according
to the affinity constants calculated by Fabiato and
Fabiato(18) . Hydrolysis at each concentration was assayed in
triplicate, and the experiment was performed three times with
independently isolated purified Ca
-ATPase. All water
used in buffers was high pressure liquid chromatography grade. For
determination of the K
for ATP, the final
concentration of ATP in the reaction was adjusted by varying the amount
of cold ATP used to make the ATP mixture. For each concentration,
hydrolysis was assayed in triplicate, and the specific activity of the
ATP was calculated and a new set of blanks obtained. The values plotted
are the average of three independent experiments. The total ATP
hydrolyzed in the assays did not exceed 6% of the total amount of
substrate present.
Inside-out membrane vesicles from F. odoratum contain at least three distinct ATPases. Approximately 25% of the
ATPase activity is sensitive to DCCD and oligomycin but not vanadate
and appears to be from a F-type
H
-ATPase(8) . The remainder of the activity is
refractory to inhibition by DCCD and oligomycin but is sensitive to
micromolar amounts of vanadate. The calcium-dependent
vanadate-sensitive ATPase activity makes up the majority of this
activity (greater than 50% of the total membrane ATPase activity). The
remaining calcium-independent vanadate-sensitive ATPase activity can be
ascribed to one or more other P-type ATPases. Thus, the
calcium-dependent ATPase activity can be easily detected, which made it
amenable to isolation.
Several detergents were tested for their
ability to solubilize the F. odoratum Ca-ATPase. The non-ionic detergent
C
E
was best, and detergents such as Triton
X-100, deoxycholate, cholate, and octyl glucoside were much less
effective. A summary of the purification scheme is shown in Table 1. Treatment of membrane vesicles with 2%
C
E
preferentially solubilizes the
calcium-dependent hydrolysis activity, leaving most of the
vanadate-sensitive calcium-independent activity and 50% of the
DCCD-sensitive F
-ATPase activity in the detergent-extracted
pellet. The weight ratio of the detergent to protein is optimized at 2%
C
E
with 18 mg/ml membrane protein. This
resulted in the solubilization of 50-70% of the calcium-dependent
vanadate-sensitive ATPase with a 2-3-fold purification. After
solubilization, proteins in the detergent extract can be sequentially
precipitated with increasing amounts of ammonium sulfate. All of the
F
-ATPase activity and 20% of the calcium-independent P-type
ATPase activity is precipitated at 55% ammonium sulfate. At 65%
ammonium sulfate saturation, the remainder of the calcium-independent
activity is precipitated. The fraction precipitated at 90% saturation
contains >95% calcium-dependent activity, which is completely
abolished by vanadate. This yields a fraction with a specific activity
of 729 nmol of ATP hydrolyzed min
mg
protein, a 30-fold purification over that in ISO membrane
vesicles and represents a little less than 50% of the
Ca
-ATPase activity in the membrane vesicles. This was
then layered over a 32-48% glycerol gradient in buffer A
containing no additional C
E
and centrifuged
180,000
g for 8 h at 4 °C. The active fractions
were pooled, concentrated, dialyzed against buffer A, and then applied
to a second 32-48% glycerol gradient and centrifuged as described
above. The active fractions were pooled and applied directly to a DE52
column. Elution is achieved with a KCl step gradient of 50 mM steps from 100 to 250 mM. The Ca
-ATPase
activity is eluted upon the addition of 200 mM KCl. The
purified Ca
-ATPase consists of a single polypeptide
component of M
60,000 that represents at least 95%
of the total protein in the final fraction (Fig. 1, lane
4). The NH
-terminal amino acid sequence was determined
by Yu Ching Pan (Roche Research Center, Nutley, NJ) and Midwest
Analytical, Inc. from both the purified preparation and the 60,000-Da
PAGE isolated band blotted onto polyvinylidene difluoride. This was to
ensure that there were no other proteins present that were not detected
by Coomassie Brilliant Blue R-25 or silver staining. As expected, both
preparations yielded identical sequences and showed there was no
significant contamination from other copurified polypeptides. The first
22 residues of the prokaryotic Ca
-ATPase are
NH
-SEKLEKPKLVVGLVVDQMRWDY. The absence of an
NH
-terminal methionine raises the distinct possibility that
the prokaryotic pump may have a leader sequence.
Figure 1:
SDS-PAGE of the active fractions from
various purification steps. Active fractions of
Ca-activated vanadate-sensitive ATPase were
dissociated and resolved by SDS-PAGE. The gel was silver stained. Lane 1, 90% ammonium sulfate pellet. Lane 2, first
glycerol density gradient fraction. Lane 3, second glycerol
density gradient fraction. Lane 4, peak fraction from DE52
chromatography.
The purified
Ca-ATPase exhibits a maximal rate of ATP hydrolysis
at pH 8.0 and is active over a narrow pH range (data not shown). The
enzyme loses 50% of its activity at pH 7.0 or 8.5 and is at less than
20% of its maximal rate at pH 6.5. It has virtually no activity at pH
9.0. The best substrate is MgATP with a K
of 90
µM (Fig. 2) and a V
of
75 µmol min
mg
. The
kinetics of hydrolysis of the purified Ca
-ATPase as a
function of [ATP] appears to be simple (hyperbolic) and
saturable. The V
of the purified prokaryotic
enzyme appears to be approximately 10-fold greater than that of the
purified SR Ca
-ATPase(22) . There is no
detectable modulatory effect of ATP on enzyme activity when examining
steady state ATP hydrolysis. Modulatory effects of ATP on the purified
calcium pump, if present, might be detectable using rapid-quench flow
techniques, which permit the resolution of individual steps in the
reaction mechanism. Fig. 3shows a Hanes-Woolf plot of
phosphointermediate (E
P) formation as a function of the
concentration of MgATP in the range of 0.5-500 µM.
Rates of E
P formation were calculated at 2-ms time points to
reflect true initial velocity at 8 °C and at 50 µM Ca
where the Ca
binding sites
would be saturated. The single turnover kinetics of E
P
(calcium) formation of the purified prokaryotic
Ca
-ATPase are simple and reveal a single K
of activation at
90 µM MgATP.
Importantly, detergent-solubilized SR Ca
-ATPase also
has simple kinetics with a single K
of MgATP
activation of 100 µM. It is only the membrane-bound form
of the SR Ca
-ATPase that has two activating phases
for ATP, one occurring between 2 and 50 µM and the other
well above 100 µM MgATP(22) . Therefore, the
kinetics observed for ATP hydrolysis and E-P formation in the purified
prokaryotic Ca
-ATPase are precisely what has been
observed for the detergent-solubilized SR Ca
-ATPase.
To determine whether the purified prokaryotic ATPase single K
for ATP is attributable to a
detergent-solubilized state like the SR pump or whether it is due to a
more fundamental difference between the two enzymes, we examined the
effect of MgATP concentration on the activation of the pump in membrane
vesicles. In contrast to what is seen in the purified ATPase, the rate
of hydrolysis for the prokaryotic pump in inside-out membrane vesicles
is a sigmoid function of MgATP concentration. A break in the
Hanes-Woolf plot (Fig. 4) was observed when the MgATP was varied
over a wide range (from 0.2 µM to 1 mM). One
straight line was observed in the range of 0.2-30 µM giving an apparent K
at 10 µM,
and a second straight line (30 µM-1 mM)
reveals an apparent K
at >400 µM.
The appearance of two K
values in this range is
consistent with what was observed for the SR
Ca
-ATPase(22) .
Figure 2:
Affinity of purified
Ca-ATPase for ATP. Initial rates of ATP hydrolysis by
the purified Ca
-ATPase were measured at varying ATP
concentrations. Blank values and specific activity were adjusted for
each new ATP concentration. Hydrolysis assays were performed in
triplicate for each point under initial rate conditions. Total ATP
hydrolyzed did not exceed 6% of the initial substrate concentration.
Rate was plotted as a function of [ATP], and the direct plot
represents an average of three independent experiments, performed on
independently isolated enzyme fractions. The data were replotted (inset) as [S]/Vversus [S], and the x intercept
(-K
) was determined by
extrapolation.
Figure 3:
E-P formation of purified
Ca-ATPase as a function of ATP concentration. Initial
rates of E-P formation by the Ca
-ATPase were examined
at 8 °C using a three-syringe rapid quench apparatus. Final
conditions were 20 mM MOPS (pH 7.0), 100 mM KCl, 1
mM MgSO
, 50 µM CaCl
, 41.7
ng/µl F. odoratum Ca
-ATPase, and ATP
concentrations as indicated. Syringe A contained varying amounts of
[
-
P]ATP (2000 cpm/pmol) at twice their
final concentration. Syringe B contained 83.3 ng/µl of
Ca
-ATPase and 100 µM CaCl
.
Syringe C contained 35% trichloroacetic acid quench solution. Other
components were present at their final concentrations in all syringes
except syringe C. Blank values and specific activity were adjusted for
each new ATP concentration. To each quenched reaction, 15 µg of
bovine serum albumin was added, and the protein was precipitated. The
precipitated protein was analyzed by SDS-PAGE. The bands were
visualized by autoradiography, cut, and then counted. The data were
plotted as [S]/V
versus [S], and the x intercept
(-K
) was determined by
extrapolation.
Figure 4:
Affinity of vesicular
Ca-ATPase for ATP. Initial rates of ATP hydrolysis by
the Ca
-ATPase in inside-out membrane vesicles were
measured at varying ATP concentrations. Blank values and specific
activity were adjusted for each new ATP concentration. Hydrolysis
assays were performed in triplicate for each point under initial rate
conditions. Total ATP hydrolyzed did not exceed 6% of the initial
substrate concentration. The data were plotted as [S]/V
versus [S], and the x intercepts
(-K
) were determined by
extrapolation.
The only other nucleotides
hydrolyzed at significant rates by the prokaryotic
Ca-ATPase are dATP, GTP, UTP, and ITP at rates of 70,
57, 25, and 25%, respectively, of that measured by ATP. ADP, CTP,
acetyl phosphate, and AMP-PNP were not hydrolyzed. P-nitrophenylphosphate, which is readily hydrolyzed by the SR
Ca
-ATPase(23) , is also hydrolyzed by the
prokaryotic Ca
pump.
Importantly, ATP hydrolysis
is absolutely dependent on Mg and
Ca
. Fig. 5shows hydrolysis of ATP by the
purified prokaryotic Ca
-ATPases as a function of pCa at an MgATP concentration of 100 µM. Similar
to the SR Ca
-ATPase, the apparent K
for calcium is 1.5 µM, and the plot of ATPase
activity at various pCa is sigmoidal (Fig. 5). The
activation process is characterized by a Hill coefficient of 2.4
(average of four experiments). This is comparable with the n =
2 value measured for the SR
Ca
-ATPase(24, 25) . Therefore, at
low concentrations of free calcium the prokaryotic
Ca
-ATPase clearly exhibits positive cooperativity for
calcium consistent with the binding of at least two calcium in its
activation just like the SR Ca
-ATPase. The addition
of 1 mM Na
, Ba
, or the
replacement of 100 mM KCl with 100 mM NaCl or with
100 mM K
SO
had little or no effect on
hydrolysis activity (data not shown). The addition of 1 mM Mn
or Cd
inhibited activity by
about 50%. Lanthanide, a calcium analog and calcium channel
inhibitor(26) , inhibits hydrolysis. Lanthanum and lanthanide
salts have an ionic radius similar to that of calcium and have been
reported to complex with ATP to form stable lanthanum
phosphointermediates in the SR Ca
-ATPase, which
dramatically inhibits its turnover rate. Significantly, no other
divalent or monovalent cation can substitute for calcium. Inesi and
colleagues (27) have clearly demonstrated that H
serves as a counter ion in the SR Ca
-ATPase
reaction mechanism. Unfortunately, we have been unsuccessful in our
attempts to reconstitute the purified F. odoratum Ca
-ATPase and therefore have not yet determined
whether it requires a H
as a counter ion.
Orthovanadate inhibits the purified prokaryotic
Ca
-ATPase in a concentration-dependent manner, with a
calculated IC
of 0.6 µM (Fig. 6). This
value is much closer to that reported for the sarcolemmal (28) and erythrocyte Ca
-ATPase of 0.5
µM(29) than the IC
of 10-µm
vanadate reported for the SR Ca
-ATPase(30) .
The purified Ca
-ATPase is insensitive to DCCD and N-ethylmaleimide. Importantly, it is also insensitive to the
SR Ca
-ATPase inhibitors fluorescein
isothiocyanate(31) , thapsigargin(32) , and
cyclopiazonic acid(33) .
Figure 5:
Calcium dependence of ATP hydrolysis of
purified Ca-ATPase. Hydrolysis was assayed using
calcium-EGTA mixtures to buffer free calcium concentration, according
to the program described by Fabiato and Fabiato(18) . ATP
concentration was maintained at 100 µM for all assays.
Hydrolysis assays were performed in triplicate for each point, under
initial rate conditions, and activity was calculated as a function of
free calcium concentration. The direct plot is an average of three
independent experiments, using independently isolated enzyme fractions.
The inset contains Hill plots of the calcium titration
curve.
Figure 6:
Inhibition by vanadate. Purified samples
were incubated for 10 min in reaction buffer of 100 mM KCl, 20
mM MOPS, pH 7.5, 0.1 mM CaCl, 0.5 mM MgSO
and vanadate at concentrations ranging from 0.1
to 100 µM. Hydrolysis assays were performed in triplicate.
Inhibition is plotted as % activity versus log
[vanadate], with an apparent IC
of 0.6
µM.
All P-type ATPases form an
acylphosphate with the -phosphate of ATP as an intermediate in the
reaction cycle. The purified Ca
-ATPase is
phosphorylated by ATP only in the presence of Ca
, and
phosphointermediate formation is inhibited by vanadate (Fig. 7A). Importantly, the phosphointermediate formed
from the purified Ca
-ATPase comigrates with the
phosphointermediate formed from F. odoratum membrane vesicles (Fig. 7B). We have also examined the reverse
phosphorylation of the purified Ca
-ATPase. We have
shown previously that unlike the SR Ca
-ATP, the
prokaryotic Ca
-ATPase is phosphorylated by P
in the presence of Ca
(10) . Fig. 7C demonstrates that E-P formation by P
in the purified Ca
-ATPase is identical to what
was seen in detergent-solubilized
Ca
-ATPase(10) . The enzyme is phosphorylated
both in the presence and absence of Ca
, and E-P
formation is much greater in the presence of Ca
. To
detect E-P formed in the absence of Ca
, the level of
purified Ca
-ATPase loaded on the gel was five times
greater than that loaded for E-P formation in the presence of
Ca
. Presumably the E
Ca-P intermediate is
less labile to hydrolysis than the E
-P intermediate.
Importantly, the phosphointermediate formed by P
in the
presence and absence of Ca
is sensitive to
hydroxylamine and inhibited by micromolar vanadate (Fig. 7D). The phosphointermediate formed in the
presence of Ca
turns over rapidly upon chase with a
500-fold excess of cold P
or ADP (Fig. 7, E and F). The phosphointermediate formed in the absence of
calcium also turns over rapidly with excess cold P
but does
not turnover in the presence of excess ADP (data not shown, see (10) ).
Figure 7:
Phosphorylation of the purified
Ca-ATPase. Forward phosphorylation (A) was
carried out on ice with 0.1 µg of purified protein incubated for 10
min in 0.1 M KCl, 20 mM MOPS, pH 7.5, and 0.5 mM MgCl
with the indicated additions of calcium (0.5
mM), EGTA (2 mM), and vanadate (0.1 mM). 20
µM [
-
P]ATP (2000 cpm/pmol) was
added to initiate the 10-s reaction, which was stopped with 750 µl
of 5% trichloroacetic acid/5 mM potassium P
. The
precipitated protein was pelleted, resuspended, and analyzed by
SDS-PAGE and fluorography. B, forward phosphorylation was
carried out on ice with 75 µg of F. odoratum, membrane
vesicles (lane 1), 0.05 µg of purified
Ca
-ATPase incubated for 10 min in 0.5 mM CaCl
, 0.1 M KCl, 20 mM MOPS, pH 7.5,
and 0.5 mM MgCl
(lane 2). 20 µM [
-
P]ATP (2000 cpm/pmol) was added to
initiate the 10-s reaction, which was stopped and analyzed for
phosphointermediate formation as described above. Reverse
phosphorylation (C-F) was carried out on ice with 0.2
µg of purified protein incubated for 10 min in 0.1 M MES,
pH 6.5, and 0.5 mM MgCl
with the indicated
additions of calcium (0.5 mM), EGTA (2 mM), and
vanadate (0.1 mM). 2 µM [
P]orthophosphate (912 Ci/mM) was
added to initiate the 10-s reaction, which was stopped with 750 µl
of 5% trichloroacetic acid, 5 mM potassium P
. The
precipitated protein was pelleted, resuspended, and analyzed by
SDS-PAGE and fluorography. In C, reactions were with the
indicated additions of calcium (0.5 mM), EGTA (2 mM),
and vanadate (0.1 mM). Total protein used in the EGTA
reactions was 1 µg. In D, all reactions included 0.5
mM CaCl
with 0.1 mM vanadate added to lane 2. In lane 3, the pellet was resuspended in 100
mM MES, pH 6.0, 100 mM hydroxylamine. In the cold
chase experiments (E and F), 0.5 mM CaCl
was included in all reaction buffers. Reactions
were initiated with the addition of 2 µM [
P]orthophosphate for 15 s, at which point
2 mM ADP (E) or 2 mM cold potassium P
(F) was added for the indicated times (0 is no chase)
before the reactions were stopped.
The SR Ca-ATPase has been extensively
characterized and serves as the structure-function model for the
analysis of ATP-catalyzed cation translocation. However, until recently
very little was known about the prokaryotic calcium pumps. This work is
the first report of the purification of a prokaryotic P-type calcium
ATPase. The isolated protein migrated as a single band on SDS-PAGE,
with an apparent M
of 60,000. Calcium-dependent
ATP hydrolysis with a V
of
75 µmol
min
mg
was fully inhibited by
micromolar vanadate, and the formation of an alkaline-labile
phosphointermediate was observed in the forward (ATP) and reverse
(P
) limbs of the reaction cycle. The K
values for calcium and ATP and the IC
for vanadate
of the purified prokaryotic ATPase are very similar to that of the SR
pump. In contrast to the SR ATPase, the prokaryotic
Ca
-ATPase is phosphorylated by P
in the
presence of calcium. This is consistent with what was observed in F. odoratum membrane vesicles and partially purified
fractions. These data suggest that either the prokaryotic
Ca
-ATPase forward reaction is ordered(10) , i.e. the enzyme does not bind calcium at the high affinity
site until ATP binds, or that ATP and not Ca
determines the direction of the reaction cycle. Significantly,
the prokaryotic enzyme's apparent M
is only
55% that of the SR Ca
-ATPase, making it the smallest
P-type ATPase reported to date. The high purification factor and final
yield of protein suggests that, though active and stable, the calcium
pump is not present in great abundance in the membrane.
The F.
odoratum calcium pump is constitutively expressed under most
growth conditions (high or low media calcium, pH, or O tension) and, unlike the derepressible Kdp ATPase, does not
appear to be subject to proteolytic degradation during purification.
The V
for the prokaryotic ATPase is at least
10-fold greater than that reported for the SR calcium pump. The
purified enzyme is very stable even at room temperature and shows no
appreciable loss of activity even with multiple freeze-thaw cycles.
Initial isolation was carried out with the addition of 0.25%
C
E
and lipids to the buffers for glycerol
gradients and DE52 chromatography. Interestingly, we later discovered
that no detergents or lipids needed to be added to any of the buffers
after the initial 2% C
E
solubilization. This
suggests that the prokaryotic Ca
-ATPase has large
hydrophilic domains that allow it to remain soluble, active, and stable
even in very low concentrations of detergents and phospholipids.
Unfortunately, so far our efforts to reconstitute the purified
prokaryotic Ca-ATPase into liposomes have not been
successful, but we are continuing to look for conditions that will
allow us to examine the calcium translocation properties of the
purified pump. Our failure to successfully reconstitute the
Ca
-ATPase brings up the possibility that we have
isolated the catalytic subunit of a multi-subunit ATPase. Importantly,
the phosphointermediate formed from the purified
Ca
-ATPase is the same size as that formed from the
membrane vesicles. Therefore, the small size, high level of turnover,
and stability in very low detergent concentrations are not due to
proteolysis of a larger more hydrophobic form. These data do support
the possibility that the 60,000-Da polypeptide is the catalytic subunit
of a larger complex and that at least a second transmembrane component
may be required for transport. The
Na
,K
-ATPase and
H
,K
-ATPase require a
subunit
for proper assembly and activity, but no homology for the
subunit
has been identified for any of the prokaryotic P-type ATPase. The
Kdp-ATPase is composed of three subunits, A, B, and C, of 60,000,
72,000, and 20,000 Da(12, 13) , respectively. KdpB
forms an acylphosphate intermediate and has regions of homology with
the eukaryotic P-type ATPases. Genetic evidence demonstrates that both
KdpA and KdpC are both required for function. None of the subunits of
the Kdp ATPase have been isolated to homogeneity or demonstrated to
have partial or complete activity. Importantly, the purified F.
odoratum Ca
-ATPase is very active, carrying out
calcium-dependent vanadate-sensitive ATP hydrolysis and forward and
reverse phosphorylation. Our attempts at reconstitution may have not
been successful because of a possible requirement for one or more of
the unusual lipids present in the F. odoratum membrane(34) . We are currently identifying conditions for
successful reconstitution and examining the possibility of other
subunits required for transport.
The purification of the
Ca-ATPase will allow us to directly address these and
other structure-function questions. Importantly, the purification of
the Ca
-ATPase has given us sequence information that
has facilitated its cloning and subsequent functional expression in E. coli. (
)The ability of heterologous expression
of mutants of the Ca
-ATPase, which can be purified
for characterization, makes this system especially useful for the
analysis of the molecular mechanisms of transport ATPases.
REFERENCES
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