(Received for publication, October 7, 1994; and in revised form, November 1, 1994)
From the
ATP-induced changes in the tryptophan fluorescence of the
Ca-ATPase were determined with sarcoplasmic reticulum
vesicles at pH 7.0 and 0 °C by steady-state measurements in the
presence of Ca
and the absence of K
with and without added lasalocid (a carboxylic ionophore, 50
µM), which was previously shown to cause a predominant
accumulation of the ADP-insensitive form of the phosphoenzyme
intermediate (EP) (Kawashima, T., Hara, H., and Kanazawa,
T.(1990) J. Biol. Chem. 265, 10993-10999). When ATP was
added in the absence of lasalocid, the fluorescence decreased by 1.7%.
The addition of lasalocid quenched 71% of the fluorescence but did not
reduce the ATP-induced fluorescence drop. The fluorescence drop and the EP formation were also determined in the presence of lasalocid
by stopped-flow spectrometry and continuous-flow rapid quenching. The
observed fluorescence drop was biphasic. The first phase coincided with
the formation of EP, which was largely ADP-sensitive in this
early stage of the reaction. The second phase was much slower than the
first phase and coincided with the accumulation of ADP-insensitive EP. When the transition of EP from the ADP-sensitive
form to the ADP-insensitive form was blocked by N-ethylmaleimide treatment, the second phase disappeared, and
the fluorescence drop entirely coincided with the formation of
ADP-sensitive EP. These findings demonstrate that the first
phase of the fluorescence drop is attributed to the formation of
ADP-sensitive EP, the second phase being attributed to the
transition of EP from the ADP-sensitive form to the
ADP-insensitive form. The present results reveal the conditions that
definitely discriminate these two phases.
The SR ()Ca
-ATPase is a 110-kDa
enzyme that is responsible for Ca
transport coupled
to ATP hydrolysis(1, 2) . According to the functional
domains predicted from its primary structure(3, 4) ,
this enzyme has the ATP-binding site (catalytic site) in the
cytoplasmic domain and the high affinity Ca
-binding
sites (transport sites) in the transmembrane domain. In the initial
step of the catalytic cycle, the enzyme is activated by Ca
binding to the transport sites from the cytoplasmic side of the
SR membrane. The
-phosphoryl group of ATP is transferred to
Asp-351 in the catalytic site of the activated enzyme (3, 5, 6, 7) to form ADP-sensitive EP, which can react with added ADP to form
ATP(8, 9, 10) . This EP formation is
accompanied by an occlusion of the bound
Ca
(11, 12, 13) . In the
subsequent conformational transition, this EP is converted to
the ADP-insensitive form. Concurrently, the affinity of the
Ca
-binding sites is greatly reduced, and the
Ca
is released into the lumen. Finally,
ADP-insensitive EP is hydrolyzed to liberate P
.
Tryptophans are useful intrinsic probes for detecting conformational
changes in the individual steps of this catalytic cycle, because their
fluorescence is sensitive to changes of the environment. In fact,
Dupont and Leigh (14) and others (15, 16, 17, 18) showed that an
addition of ATP to the Ca-activated enzyme induces a
tryptophan fluorescence drop. However, in those studies the assignment
of this fluorescence drop to a specific reaction step is not
definitive. Recently, we have shown (19) that the tryptophan
fluorescence remains unchanged when the calcium-activated enzyme-ATP
complex is formed and that the ATP-induced tryptophan fluorescence drop
reflects a conformational change upon formation of ADP-sensitive EP in the presence of K
, which accelerates
hydrolysis of ADP-insensitive EP and suppresses its
accumulation(20) . For a better understanding of the molecular
mechanism of Ca
transport, it is important to examine
whether the tryptophan fluorescence change occurs on the transition of EP from the ADP-sensitive form to the ADP-insensitive form.
However, attempts to detect the tryptophan fluorescence change on this
transition in the absence of K
were unsuccessful
because of the insufficient accumulation of ADP-insensitive EP.
In this study, by stopped-flow spectrofluorometry and
continuous-flow rapid quenching, we have determined the ATP-induced
tryptophan fluorescence change and EP formation in the
presence of Ca and the absence of K
with added lasalocid (a carboxylic ionophore), which was
previously shown (21) to inhibit hydrolysis of ADP-insensitive EP and to cause a predominant accumulation of this EP. The results show that the tryptophan fluorescence drop on
the transition of EP from the ADP-sensitive form to the
ADP-insensitive form is revealed under these conditions.
Figure 1:
Effects of lasalocid and A23187 on
ATP-induced fluorescence drop. A, the measurement of
steady-state fluorescence was started at 0 °C with 2.39 ml of a
suspension of SR vesicles, and then 12 µl of ethanol (left
trace) or 10 mM lasalocid dissolved in ethanol (center and right traces) was added. The resulting
mixture had a composition of 0.05 mg/ml SR vesicles, 10 mM MgCl, 0.55 mM CaCl
, 0.5 mM EGTA (55 µM free Ca
), 0.1 M LiCl, 20 mM MOPS/Tris (pH 7.0), 0.5% ethanol, and 50
µM lasalocid (center and right traces only). In the left and right traces, 0.74 µl
of 98 mM ATP was added 30 min after the addition of ethanol or
lasalocid. B, the measurement was started as in A,
and then 24 µl of ethanol (left trace) or 3 mM A23187 dissolved in ethanol (center and right
traces) was added. The resulting mixture had the same composition
as in A except for 1% ethanol (all traces) and 30
µM A23187 in place of 50 µM lasalocid (center and right traces only). In the left and right traces, 0.74 µl of 98 mM ATP was
added 30 min after the addition of ethanol or A23187. In the left
traces of A and B, the fluorescence intensity
before the addition of ATP is normalized to 100%. In the center and right traces of A and B, the
fluorescence intensity before the addition of lasalocid or A23187 is
normalized to 100%. The right traces of A and B show the data only after the addition of lasalocid or
A23187.
The fluorescence was also markedly
quenched by 30 µM A23187, the residual fluorescence being
only 3% (center trace in Fig. 1B). However, in
contrast to the results obtained with lasalocid, the ATP-induced
fluorescence drop was almost completely inhibited by A23187, which was
added previously (right trace in Fig. 1B).
This finding is in agreement with our recent results(19) ,
which were obtained in the presence of 100 mM K and 0.25 mM CaCl
without added MgCl
and EGTA under otherwise similar conditions. It should be noted
that A23187 at the high concentration used in this experiment does not
inhibit the formation of ADP-sensitive EP, although it
inhibits the transition of EP from the ADP-sensitive form to
the ADP-insensitive form(27) . Thus, the present findings
indicate that tryptophans responsible for the fluorescence drop upon
formation of ADP-sensitive EP are highly susceptible to the
fluorescence energy transfer to the added A23187.
It was previously
suggested that A23187 (18, 28) and lasalocid (26) preferentially quench the fluorescence of tryptophans in
or near the transmembrane domain of the Ca-ATPase by
binding to the protein-lipid interface of this enzyme. It is likely
that tryptophans responsible for the ATP-induced fluorescence drop are
located in or near the transmembrane domain, because the ATP-induced
fluorescence drop was completely inhibited by A23187 (Fig. 1B). The lack of effect of lasalocid on the
ATP-induced fluorescence drop (Fig. 1A) may be due to
the binding of lasalocid to a site(s) far distant from the tryptophans
or due to the difference in the Förster distance
(including the difference in the orientation factor) between A23187 and
lasalocid.
Figure 2:
Time
courses of ATP (4 µM)-induced fluorescence drop and
accumulation of ADP-insensitive EP. SR vesicles (0.05 mg/ml)
were preincubated with 50 µM lasalocid at 0 °C for 2 h
in a medium containing 10 mM MgCl, 0.55 mM CaCl
, 0.5 mM EGTA (55 µM free
Ca
), 0.1 M LiCl, and 20 mM MOPS/Tris (pH 7.0) and then mixed with 8 µM ATP in
the same medium at 0 °C by the stopped-flow method. The reaction
was followed for 30 (A) or 3 (B) s. The ATP-induced
fluorescence change (small filled circles) was obtained by
subtracting the base-line level, which was determined by mixing the
preincubated vesicles with the ATP-free medium. The fluorescence change
was biphasic. In A, a part of the first phase was completed
within the instrumental dead time. The second phase was fitted to a
single exponential (middle solid line), which gave a
first-order rate constant of 0.42 s
and an amplitude
of 22.0 units on a relative scale. In B, the fluorescence
change was fitted to two exponentials (middle solid line). The
first exponential gave a first-order rate constant of 5.8
s
and an amplitude of 25.4 units, and the second
exponential gave a first-order rate constant of 0.43 s
and an amplitude of 19.0 units. EP formation was
performed with [
-
P]ATP by the
continuous-flow rapid quenching method or by manual pipetting under
conditions that were otherwise similar to those in the stopped-flow
measurement, and the total amount of EP (
) and the amount
of ADP-insensitive EP (
) were determined. The top
solid lines in A and B show the best fit of a
single exponential to the time course of EP formation, in
which the first-order rate constant and the maximum of the total amount
of EP were 3.5 s
and 2.90 nmol/mg,
respectively. The bottom solid lines in A and B show the best fit of a single exponential to the time course of
the accumulation of ADP-insensitive EP, in which the
first-order rate constant and the maximum amount of ADP-insensitive EP were 0.28 s
and 2.53 nmol/mg,
respectively.
The first phase of the ATP-induced fluorescence drop coincided fairly well with EP formation (Fig. 2B). In this early stage of the reaction, most of the EP formed was ADP-sensitive. Thus, this finding is consistent with our previous observations (19) that the ATP-induced tryptophan fluorescence drop is attributed to the formation of ADP-sensitive EP under the conditions where ADP-sensitive EP accumulates almost exclusively.
The second phase of the ATP-induced fluorescence drop appeared to coincide with the accumulation of ADP-insensitive EP. This probability was supported by the further experiment in which ATP was added to give 50 µM (Fig. 3). At this higher concentration of ATP, the first phase of the fluorescence drop and EP formation were very rapid and completed within the instrumental dead times. This made it possible to compare the second phase of the fluorescence drop with the accumulation of ADP-insensitive EP without interference from the first phase of the fluorescence drop. The results clearly show that the second phase of the fluorescence drop coincided with the accumulation of ADP-insensitive EP and was well fitted with a single exponential.
Figure 3:
Coincidence between the second phase of
ATP (50 µM)-induced fluorescence drop and the accumulation
of ADP-insensitive EP. The SR vesicles preincubated with
lasalocid were mixed with 100 µM ATP, under the conditions
described in the legend of Fig. 2. The ATP-induced fluorescence
change (small filled circles) was obtained by subtracting the
base-line level as in Fig. 2. Because the first phase of the
fluorescence change finished within the instrumental dead time, the
data represent only the second phase of the fluorescence change. The lower solid line shows the best fit of a single exponential to
the second phase of the fluorescence change, in which the first-order
rate constant and the amplitude were 0.38 s and 20.1
units, respectively. EP formation was performed with
[
-
P]ATP under conditions that were
otherwise similar to those in the stopped-flow measurement, and the
total amount of EP (
) and the amount of ADP-insensitive EP (
) were determined.
Figure 4:
Lack of the second phase of ATP-induced
fluorescence drop and inhibition of the transition of EP from
ADP-sensitive form to ADP-insensitive form in NEM-treated SR vesicles.
The experiment was performed with NEM-treated SR vesicles under the
conditions described in the legend of Fig. 2, and the
ATP-induced fluorescence change (small filled circles), the
total amount of EP (), and the amount of ADP-insensitive EP (
) were determined. The reaction was followed for 30 (A) or 3 (B) s. The solid lines in A show spline fits. The upper solid line in B shows the best fit of a single exponential to the fluorescence
change, in which the first-order rate constant and the amplitude were
5.7 s
and 22.7 units, respectively. The lower
solid line in B shows the best fit of a single
exponential to the time course of EP formation, in which the
first-order rate constant and the maximum of the total amount of EP were 4.0 s
and 2.38 nmol/mg,
respectively.
The rate of EP formation was little affected, and the maximum of the total amount of EP was only slightly reduced by this treatment, whereas the accumulation of ADP-insensitive EP was almost entirely suppressed. These results show that the transition of EP from the ADP-sensitive form to the ADP-insensitive form was almost completely inhibited by the NEM treatment, being consistent with our previous findings(19, 29) , which were obtained in the absence of lasalocid under otherwise similar conditions.
The ATP-induced fluorescence drop was monophasic and well fitted with a single exponential (Fig. 4B), being in sharp contrast to the biphasic fluorescence drop observed with the untreated vesicles (Fig. 2). The rate and amplitude of the fluorescence drop were essentially the same as those of the first phase of the fluorescence drop with the untreated vesicles (Fig. 2B). Thus, the ATP-induced fluorescence drop with the NEM-treated vesicles was entirely devoid of the second phase.
The fluorescence drop coincided fairly well with the formation of EP, almost all of which was ADP-sensitive. This finding adds support to the preceding conclusion (Fig. 2) that the first phase of the ATP-induced fluorescence drop is attributed to the formation of ADP-sensitive EP. Furthermore, the lack of the second phase of the fluorescence drop, accompanied by the almost complete inhibition of the transition of EP, gives evidence that the second phase of the ATP-induced fluorescence drop is attributed to the transition of EP from the ADP-sensitive form to the ADP-insensitive form. In harmony with this conclusion, Fernandez-Belda et al.(16) and Champeil et al.(18) suggest that the ATP-induced fluorescence drop predominantly monitors the transition of EP. However, because they did not determine ADP-insensitive EP, it is not known whether ADP-insensitive EP actually accumulated in those experiments.
The present findings, together with our recent
observations(19) , have revealed the experimental conditions
that definitely discriminate the two phases of the ATP-induced
fluorescence drop. The first phase is selectively observed in the
presence of K and the absence of
lasalocid(19) , whereas the second phase is in the absence of
K
and the presence of lasalocid at rather high ATP
concentrations (Fig. 3).
It appears likely that the first
phase of the fluorescence drop reflects a conformational change
associated with Ca occlusion, which is coupled with
the formation of ADP-sensitive EP, and that the second phase
reflects a conformational change associated with Ca
release, which is coupled with the transition of EP from
the ADP-sensitive form to the ADP-insensitive form. The discrimination
of these two phases may be useful for the detailed investigation of
conformational events in these steps of the Ca
transport.
We previously showed that conformational changes
occur in the catalytic site of the cytoplasmic domain in three
successive steps: formation of the Ca-activated
enzyme-ATP complex, formation of ADP-sensitive EP, and
transition of EP from the ADP-sensitive form to the
ADP-insensitive form(24, 29, 30) . It is
likely that the conformational changes in the cytoplasmic domain are
transmitted to the Ca
-binding sites in the
transmembrane domain only upon the latter two steps, because our recent
study (19) has shown that no change in the tryptophan
fluorescence is detected upon the formation of the
Ca
-activated enzyme-ATP complex although 12 of the 13
tryptophans of this enzyme are located in or near the transmembrane
domain(3, 4) .
where EATP, E
P, and E
P denote the calcium-activated enzyme-ATP
complex, ADP-sensitive EP, and ADP-insensitive EP,
respectively. k
and k
are
first-order rate constants. The fluorescence change (
F)
at time t is represented by ,
where
F
and
F
denote the
differences in the fluorescence intensity between E
ATP
and E
P and between E
P and E
P, respectively. When k
k
and
k
F
k
F
,
this equation is reduced to
F =
F
(1-exp(-k
t))
+
F
(1-exp(-k
t)).