©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Tryptophan Fluorescence Change upon Conformational Transition of the Phosphoenzyme Intermediate in Sarcoplasmic Reticulum Ca-ATPase Is Revealed in the Absence of K and the Presence of Lasalocid (*)

(Received for publication, October 7, 1994; and in revised form, November 1, 1994)

Hiroshi Suzuki Tohru Kanazawa (§)

From the Department of Biochemistry, Asahikawa Medical College, Asahikawa 078, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

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.


INTRODUCTION

The SR (^1)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(i).

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.


EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles

SR vesicles were prepared from rabbit skeletal muscle and stored at -80 °C as described previously (19) . The content of the phosphorylation site in this preparation was 4.13 ± 0.07 nmol/mg (n = 5) when determined with [-P]ATP according to Barrabin et al.(22) .

Treatment of SR Vesicles with NEM

In some experiments, the SR vesicles were treated with NEM in the presence of AMP-P(NH)P according to Kawakita et al.(23) . The content of the phosphorylation site was unaffected by this treatment.

Steady-state Measurements of Tryptophan Fluorescence

The steady-state intensity of the tryptophan fluorescence of SR vesicles was measured on a computer-interfaced spectrofluorometer as described previously(24) . The excitation and emission wavelengths were set to 290.0 and 338.4 nm, respectively. Slits that were 1.5 and 5.0 nm in width were set in the excitation and emission light paths.

Stopped-flow Measurements of Tryptophan Fluorescence

Rapid kinetic measurements of the tryptophan fluorescence were made by using a stopped-flow spectrofluorometer interfaced with a personal computer which was programmed to accumulate the digitized data, as described previously(19) . The excitation wavelength was 290 nm. The emitted light was passed through a band-pass filter UV-D36A (Toshiba) which cut off the light below 308 nm and above 410 nm in order to avoid interference from the fluorescence of lasalocid. The measurement was repeated 400-800 times, and the accumulated data were analyzed by the nonlinear least square method as described(19) .

Determination of EP

Continuous-flow rapid quenching measurements of EP formation were made as described previously(19) . For the determination of the total amount of EP, the reaction was started by mixing equal volumes of solutions from two syringes, one containing SR vesicles and lasalocid in a medium and the other containing [-P]ATP in the same medium. The reaction was quenched with trichloroacetic acid containing carrier ATP and P(i), and the amount of EP was determined as described previously(19) . For the determination of the amount of ADP-insensitive EP, the reaction was started as described above, and at different times thereafter an equal volume of an ADP/EGTA mixture in the same medium as above was added from the third syringe to give 5 mM ADP and 5 mM EGTA. At 100-580 ms after this addition, the reaction was quenched by spouting the reaction mixture into a cuvette containing the trichloroacetic acid, and the amount of EP remaining was determined. ADP-sensitive EP disappeared within 100 ms after the addition of the ADP/EGTA mixture, whereas ADP-insensitive EP was not significantly decomposed because of the lasalocid-induced inhibition of hydrolysis of ADP-insensitive EP(21) . When the reaction was long enough, the above procedures were carried out by manual pipetting.

Miscellaneous Methods

Protein concentrations were determined by the method of Lowry et al.(25) with bovine serum albumin as a standard. Lasalocid was purchased from Sigma. Other methods were as described previously(19) .


RESULTS AND DISCUSSION

Effects of Lasalocid and A23187 on ATP-induced Fluorescence Drop

The effects of lasalocid and A23187, which are lipophilic ionophores, on the ATP-induced fluorescence change were investigated without K in the presence of 10 mM MgCl(2) and 55 µM Ca (Fig. 1). When the ionophores were absent, an addition of ATP at saturating 30 µM(19) induced a fluorescence drop by 1.7% (left traces in Fig. 1, A and B). When lasalocid was added to give 50 µM, 71% of the fluorescence was quenched (center trace in Fig. 1A). This is consistent with the previously reported findings (26) that the tryptophan fluorescence of the SR Ca-ATPase is strongly quenched by lasalocid through the fluorescence energy transfer. When ATP was added in the presence of lasalocid, the fluorescence decreased by 2.0% of that obtained before the addition of lasalocid (right trace in Fig. 1A). Thus, the amplitude of the ATP-induced fluorescence drop was not reduced by the addition of lasalocid, although the total fluorescence was greatly quenched. These results indicate that tryptophans responsible for the ATP-induced fluorescence drop are not susceptible to the fluorescence energy transfer to the added lasalocid.


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(2), 0.55 mM CaCl(2), 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(2) without added MgCl(2) 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.

Coincidence between the Second Phase of ATP-induced Fluorescence Drop and the Accumulation of ADP-insensitive EP

SR vesicles were preincubated with 50 µM lasalocid in the presence of 10 mM MgCl(2) and 55 µM Ca without K and then mixed with ATP. The ATP-induced fluorescence drop (right trace of Fig. 1A) and EP formation were followed by stopped-flow spectrofluorometry and continuous-flow rapid quenching, respectively. When ATP was added to give 4 µM, it was found that the fluorescence drop was biphasic and fitted well with two exponentials (Fig. 2). (^2)The first phase of the fluorescence drop was much faster than the second phase, and the amplitude of the first phase was somewhat larger than that of the second phase. The total amount of EP increased rapidly after the addition of ATP and reached a steady-state level in about 1 s. The transition of EP from the ADP-sensitive form to the ADP-insensitive form was much slower, and the amount of ADP-insensitive EP reached a steady-state level in about 15 s (Fig. 2A). The accumulated ADP-insensitive EP amounted to 87% of the maximum of the total amount of EP. This predominant accumulation of ADP-insensitive EP is consistent with our previous observations (21) that hydrolysis of ADP-insensitive EP is selectively inhibited by lasalocid at the high concentration used in the present experiments.


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(2), 0.55 mM CaCl(2), 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 (circle) and the amount of ADP-insensitive EP (bullet) 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 (bullet) and the amount of ADP-insensitive EP (circle) were determined.



Lack of the Second Phase of ATP-induced Fluorescence Drop and Inhibition of the Transition of EP from the ADP-sensitive Form to the ADP-insensitive Form in NEM-treated SR Vesicles

In order to obtain further evidence to show 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, SR vesicles were pretreated with NEM under the conditions in which this transition should be selectively blocked(23) . The ATP-induced fluorescence drop and EP formation were followed with the NEM-treated vesicles under the same conditions as described in the legend of Fig. 2(Fig. 4).


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 (circle), and the amount of ADP-insensitive EP (bullet) 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) .


FOOTNOTES

*
This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, and Culture, Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, Asahikawa Medical College, Nishikagura, Asahikawa 078, Japan. Fax: 81-166-66-2635.

(^1)
The abbreviations used are: SR, sarcoplasmic reticulum; EP, phosphoenzyme; MOPS, 3-(N-morpholino)propanesulfonic acid; NEM, N-ethylmaleimide; AMP-P(NH)P, adenyl-5`-yl imidodiphosphate.

(^2)
Fitting two exponentials is rationalized as follows.

where EbulletATP, E(1)P, and E(2)P denote the calcium-activated enzyme-ATP complex, ADP-sensitive EP, and ADP-insensitive EP, respectively. k(1) and k(2) are first-order rate constants. The fluorescence change (DeltaF) at time t is represented by ,

where DeltaF(1) and DeltaF(2) denote the differences in the fluorescence intensity between EbulletATP and E(1)P and between E(1)P and E(2)P, respectively. When k(1)k(2) and k(1)bulletDeltaF(1)k(2)bulletDeltaF(2), this equation is reduced to DeltaF = DeltaF(1)(1-exp(-k(1)t)) + DeltaF(2)(1-exp(-k(2)t)).


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