Stoichiometry of Phosphorylation to Fluorescein 5-Isothiocyanate Binding in the Ca2+-ATPase of Sarcoplasmic Reticulum Vesicles*

(Received for publication, September 18, 1996)

Satoshi Nakamura Dagger §, Hiroshi Suzuki Dagger and Tohru Kanazawa Dagger

From the Departments of Dagger  Biochemistry and § Dermatology, Asahikawa Medical College, Asahikawa 078, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

In an attempt to establish the stoichiometry of phosphorylation in the Ca2+-ATPase of sarcoplasmic reticulum (SR) vesicles, phosphorylation by ATP (or Pi) or labeling by fluorescein 5-isothiocyanate (FITC) was performed with the SR vesicles under the conditions in which almost all the phosphorylation sites or FITC binding sites are phosphorylated or labeled. The resulting vesicles were solubilized in lithium dodecyl sulfate and then the Ca2+-ATPase was purified by size exclusion high performance liquid chromatography. Peptide mapping and sequencing of the tryptic digest of the purified enzyme showed that Lys-515 of the Ca2+-ATPase was exclusively labeled with FITC, in agreement with the previously reported findings. The content of the phosphoenzyme from ATP (4.57 nmol/mg of Ca2+-ATPase protein) or from Pi (4.94 nmol/mg of Ca2+-ATPase protein) in the purified enzyme was approximately half the content of the FITC binding site (8.17-8.25 nmol/mg of Ca2+-ATPase protein) and also half the content of the Ca2+-ATPase molecule (9.06 nmol/mg of Ca2+-ATPase protein) calculated from its molecular mass (110,331 Da). These results show that there is one specific FITC binding site per molecule of the Ca2+-ATPase (in agreement with the previously reported findings) and that the stoichiometry of phosphorylation to FITC binding is approximately 0.5:1.0. All the above findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated. FITC binding completely inhibited the ATP-induced phosphorylation before the binding reached its maximum level. This finding indicates that FITC preferentially binds to a part of the Ca2+-ATPase molecules and that this binding is primarily responsible for the inhibition of phosphorylation, suggesting an intermolecular ATPase-ATPase interaction.


INTRODUCTION

The SR1 Ca2+-ATPase consists of a single 110-kDa polypeptide chain, of which the whole amino acid sequence has been revealed (1). In the early step of the catalytic cycle, the gamma -phosphoryl group of Mg·ATP bound to the Ca2+-activated enzyme is transferred to Asp-351 (2, 3) to form an acid-stable EP intermediate (4, 5). EP can also be formed from Pi by reversal of the late step of the catalytic cycle (6, 7).

We have recently shown that, when SR vesicles are treated with fluoride in the presence of Mg2+, two Mg2+ and four F- per phosphorylation site bind tightly to the catalytic site of the Ca2+-ATPase (8). This finding and the related kinetic data obtained previously (9) have suggested that only half of the Ca2+-ATPase molecules present in the SR vesicles are catalytically active (i.e. the active molecules can be phosphorylated with ATP or Pi), the other half being catalytically inactive (i.e. the inactive molecules cannot be phosphorylated with ATP or Pi), and that one Mg2+ and two F- bind equally to each of the catalytically active and inactive molecules. In the subsequent study (10), we have determined the total content of the sulfhydryl group of the Ca2+-ATPase in the SR vesicles after exhaustive reduction with dithiothreitol and found that the content of the phosphorylation site in the SR vesicles is again half that of the Ca2+-ATPase molecule calculated from this sulfhydryl content.

It is now important to establish the stoichiometry of phosphorylation in the Ca2+-ATPase of SR vesicles, because the information on this stoichiometry is essential for the construction of the mechanistic model of this enzyme. A feasible approach to this problem is to determine the binding of FITC to this enzyme, because specific binding of FITC to Lys-515 at or near the ATP binding site of this enzyme is well established (11, 12).

In this study, phosphorylation by ATP (or Pi) or labeling by FITC has been performed with the SR vesicles under the conditions in which almost all the phosphorylation sites or FITC binding sites are phosphorylated or labeled. The results show that there is one specific FITC binding site per molecule of the Ca2+-ATPase (in agreement with the previously reported findings (12)) and that the stoichiometry of phosphorylation to FITC binding is approximately 0.5:1.0. These findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated.


EXPERIMENTAL PROCEDURES

Preparation of SR Vesicles

SR vesicles were prepared from rabbit skeletal muscle as described previously (8) and stored in 0.3 M sucrose, 0.1 mM CaCl2, 0.1 M KCl, and 5 mM MOPS/Tris (pH 7.0) at -80 °C.

Labeling of SR Vesicles with FITC

FITC was freshly dissolved in N,N-dimethylformamide. The SR vesicles (0.5 mg of protein/ml) were labeled with 15 µM FITC at 25 °C in the dark in 2 mM EGTA, 5 mM MgCl2, 0.1 M KCl, 1% (v/v) N,N-dimethylformamide, and 50 mM Tris/HCl (pH 8.0), unless otherwise stated. The FITC-labeled vesicles were isolated by either of the following methods. Method A: the labeling was stopped by adding 4.5 volumes of an ice-cold solution containing 12.2 mM ATP, 2 mM EGTA, 5 mM MgCl2, 0.1 M KCl, and 122 mM MOPS/Tris (pH 7.0). The resulting suspension was centrifuged at 411,000 × g and 0 °C for 5 min. Method B: the labeling was stopped at 4 °C by adding 200 mM ATP and 200 mM MES/Tris (pH 6.0) to give 10 and 36 mM, respectively. The resulting suspension was applied at 4 °C to a coarse Sephadex G-50 column preequilibrated with 0.5 mM EGTA, 0.1 M KCl, 0.3 M sucrose, and 2.5 mM MOPS/Tris (pH 7.0). The FITC-labeled vesicles in the effluent were collected by centrifugation as in Method A. 

Size Exclusion HPLC of SR Vesicles Labeled with FITC and Solubilized in LDS

The FITC-labeled SR vesicles (5 mg of protein/ml) were solubilized at 4 °C in a buffer containing 2% (w/v) LDS, 0.1 mM CaCl2, 5 mM MgCl2, 100 mM Li2SO4, and 20 mM sodium phosphate (pH 7.0). After centrifugation at 0 °C, 0.3-0.4 ml of the supernatant was subjected to size exclusion HPLC at room temperature by the use of a TSK SWXL guard column (0.6 × 4 cm, Tosoh, Japan), a TSKgel G3000SWXL column (0.78 × 30 cm, Tosoh, Japan), and a TSKgel G3000SW column (0.75 × 30 cm, Tosoh, Japan) that were connected in series. The elution was performed at a flow rate of 0.5 ml/min (unless otherwise stated) with the above solubilization buffer. The absorbance (280 nm) and fluorescence (excitation at 485 nm and emission at 525 nm) were monitored with a detector UV-M (Pharmacia Biotech Inc.) and a fluorescence detector F-1000 (Hitachi, Japan), respectively. The fractions of 0.25 ml were collected.

Determination of Bound FITC

The contents of FITC in the above fractions were determined from the absorbance at 496 nm in 8% (w/v) SDS and 200 mM Tris/HCl (pH 8.8) by using an extinction coefficient of 80,000, which was widely used in other laboratories (12-16).

Tryptic Digestion of the FITC-labeled Ca2+-ATPase and Reversed Phase HPLC

The fraction at the peak of protein concentration in the size exclusion HPLC was applied twice to centrifuge columns (5-ml disposable syringe filled with coarse Sephadex G-50) preequilibrated with 1 mM CaCl2 and 10 mM sodium phosphate (pH 7.0) at 4 °C according to Penefsky (17) in order to remove excess dodecyl sulfate. The FITC-labeled Ca2+-ATPase thus obtained was digested with trypsin (1.4:1 of trypsin to ATPase by weight) at 37 °C for 10 min in 1 mM CaCl2 and 10 mM sodium phosphate (pH 7.0). Reaction was stopped by addition of a 2-fold excess of trypsin inhibitor. The supernatant obtained by centrifugation was subjected to reversed phase HPLC, which was performed at a flow rate of 1 ml/min by using a C2/C18 SuperPac Pep-S column (5 µm, 0.4 × 25 cm, Pharmacia Biotech Inc.). The absorbance was monitored at 214 nm with a detector SPD-10AV (Shimadzu, Japan) and the fluorescence monitored in the same way as in the size exclusion HPLC.

Sequencing and Amino Acid Analysis of Isolated Peptides

Sequencing of isolated peptides was performed with a protein sequenator 477A (PE Applied Biosystems) connected with a phenylthiohydantoin-derivative analyzer 120A (PE Applied Biosystems). Amino acid analysis was performed by the use of an amino acid analyzer 835-10 (Hitachi, Japan) after hydrolysis of the peptides in 6 N HCl at 110 °C for 24 h.

Phosphorylation of SR Vesicles with ATP or Pi

Phosphorylation was performed under the conditions of Barrabin et al. (18), in which almost all the phosphorylation sites of the Ca2+-ATPase can be phosphorylated with ATP or Pi. When EP was formed from ATP, the SR vesicles (1 mg of protein/ml) were phosphorylated with 0.1 mM [gamma -32P]ATP at 25 °C for 5 s in 10 mM CaCl2, 5 mM MgCl2, 80 mM KCl, and 30 mM Tris/HCl (pH 7.5). The reaction was quenched with 9 volumes of ice-cold 5.6% (w/v) trichloroacetic acid containing 0.56 mM ATP and 5.6 mM Pi. The resulting mixture was centrifuged at 0 °C, and the pellet was washed at 4 °C once with distilled water (unless otherwise stated). When EP was formed from Pi, the SR vesicles (1 mg of protein/ml) were phosphorylated with 2 mM 32Pi at 25 °C for 10 min in 1 mM EGTA, 20 mM MgCl2, 40% (v/v) Me2SO, and 30 mM MES/Tris (pH 6.0). The reaction was quenched with 9 volumes of ice-cold 5.6% (w/v) trichloroacetic acid containing 5.6 mM PPi and 111 mM Pi. The pellet was washed at 4 °C twice (unless otherwise stated) with 4% (v/v) perchloric acid containing 20 mM Pi and 5 mM PPi and then once with distilled water.

Size Exclusion HPLC of SR Vesicles Phosphorylated and Solubilized in LDS

The phosphorylated SR vesicles (5 mg of protein/ml) were solubilized at 4 °C in a buffer containing 2% (w/v) LDS, 100 mM Li2SO4, and 50 mM lithium acetate (pH 4.5). After centrifugation at 0 °C, 0.3-0.4 ml of the supernatant was subjected to the size exclusion HPLC described above at room temperature. The elution was performed at a flow rate of 0.5 ml/min with the above acidic solubilization buffer (pH 4.5), and the absorbance was monitored at 280 nm. The fractions of 0.25 ml were collected. The radioactivity of each fraction was measured with Cerenkov radiation using a liquid scintillation counter. The content of EP was obtained from the radioactivity and protein concentration.

Miscellaneous Methods

FITC, trypsin-treated with L-1-tosylamide-2-phenylethyl chloromethyl ketone, and soybean trypsin inhibitor (type I-S) were purchased from Sigma. [gamma -32P]ATP was prepared by the method of Post and Sen (19). 32Pi was purified according to Kanazawa and Boyer (6). Protein concentrations were determined by the method of Lowry et al. (20) with bovine serum albumin as a standard.


RESULTS

Size Exclusion HPLC of SR Vesicles Labeled with FITC and Solubilized in LDS

Fig. 1 shows the size exclusion HPLC of the SR vesicles which were labeled with FITC for 200 min (FITC labeling reached almost the highest level; see the inset of Fig. 5) and solubilized in LDS. The major peak of the absorbance at the retention time of 12 min (Fig. 1A) was highly enriched with 110-kDa chains derived from the Ca2+-ATPase, as revealed by SDS-polyacrylamide gel electrophoresis (not shown). Several minor peaks of lower molecular mass proteins eluted after the major peak may be mostly assigned to the well documented Ca2+-binding proteins (21). The content of FITC per mg of protein was constant throughout six successive fractions in the major peak of proteins (Fig. 1B). A small amount of residual unreacted FITC was eluted at the retention time of 26 min.


Fig. 1. Size exclusion HPLC of SR vesicles labeled with FITC and solubilized in LDS. The SR vesicles were labeled with FITC for 200 min, and the labeling was stopped by Method A. The labeled vesicles were solubilized in LDS, applied to size exclusion HPLC, and eluted at a flow rate of 1 ml/min. The protein concentration and FITC content in each fraction of the major peak were determined. A, absorbance (2-mm optical path length) and protein concentration. B, fluorescence of FITC and content of bound FITC.
[View Larger Version of this Image (18K GIF file)]



Fig. 5. Inhibition of ATP-induced phosphorylation by FITC binding. The SR vesicles were labeled with FITC for various periods. The labeled vesicles were isolated by Method B and suspended in 0.1 mM CaCl2, 50 mM NaCl, 0.3 M sucrose, and 5 mM MOPS/Tris (pH 7.0). The vesicles in one-half of the suspension were solubilized in LDS and applied to size exclusion HPLC. The FITC content at the peak of protein concentration was determined. The vesicles in the other half of the suspension was phosphorylated with [gamma -32P]ATP, solubilized in LDS, and applied to size exclusion HPLC. The EP content at the peak of protein concentration was determined and plotted versus the FITC content. In the inset, the contents of FITC and EP were plotted versus the period of FITC labeling.
[View Larger Version of this Image (17K GIF file)]


The fraction containing 8.87 nmol of FITC/mg of protein at the peak of protein concentration was applied to the second size exclusion HPLC with the same column system as in the first size exclusion HPLC. The elution profile at 280 nm showed a single symmetric peak (not shown). The content of FITC was constant throughout seven successive fractions in this peak, being 8.43-8.96 nmol of FITC/mg of protein. This agreement in the FITC content between the first and second size exclusion HPLC indicates that the Ca2+-ATPase protein eluted in the major peak in the first size exclusion HPLC is sufficiently pure for the determination of FITC bound to the Ca2+-ATPase.

Reversed Phase HPLC of the Tryptic Digest of FITC-labeled Ca2+-ATPase

The FITC-labeled Ca2+-ATPase purified by the first size exclusion HPLC was digested with trypsin. After centrifugation of the digest, 90% of bound FITC was recovered in the supernatant. This supernatant was applied to reversed phase HPLC (Fig. 2). The fluorescence profile showed one minor peak (Peak 1) and the other major peak (Peak 2).


Fig. 2. Reversed phase HPLC of the tryptic digest of the Ca2+-ATPase purified from FITC-labeled SR vesicles. The SR vesicles were labeled with FITC for 240 min, and the labeling was stopped by Method A. The labeled vesicles were solubilized in LDS and applied to size exclusion HPLC. The fraction at the peak of protein concentration was subjected to tryptic digestion and applied to reversed phase HPLC. The elution was performed with the following linear gradient of acetonitrile in 5 mM sodium phosphate (pH 6.9) and 20 mM Na2SO4; 0% from 0 to 10 min, 9% at 20 min, 30% at 90 min, and 60% from 100 min to the end of the run. A, fluorescence of FITC. B, absorbance of peptides.
[View Larger Version of this Image (19K GIF file)]


Sequence Analysis

Peak 1 and Peak 2 in the reversed phase HPLC were further purified repeatedly by reversed phase HPLC. The fluorescence profiles in these repeated HPLC showed that fluorescent components of Peak 1 and Peak 2 were both homogeneous (not shown). Sequencing of purified Peak 1 gave no phenylthiohydantoin-derivatives. Amino acid analysis of this peak gave no amino acids, being consistent with the results of sequencing. Sequencing of purified Peak 2 gave a single sequence from Met-512 to Arg-524 of the Ca2+-ATPase (1), but the Lys-515 residue was missing. This indicates that Lys-515 was labeled with FITC. Amino acid analysis of this peak showed the composition expected from the above sequence.

Size Exclusion HPLC of SR Vesicles Phosphorylated and Solubilized in LDS

The SR vesicles phosphorylated with [gamma -32P]ATP and solubilized in LDS were subjected to size exclusion HPLC (Fig. 3), in which an acidic solubilization-elution buffer (pH 4.5) was used to stabilize EP and to facilitate its quantitative recovery. The major peak of the absorbance at the retention time of 24 min was highly enriched with 110-kDa chains derived from the Ca2+-ATPase, as revealed by SDS-polyacrylamide gel electrophoresis (not shown). The content of EP per mg of protein was constant throughout five successive fractions in this peak. Some radioactivities eluted at the retention time of 50 min are most likely attributed to residual [gamma -32P]ATP and 32Pi.


Fig. 3. Size exclusion HPLC of SR vesicles phosphorylated with ATP and solubilized in LDS. The SR vesicles were phosphorylated with [gamma -32P]ATP, solubilized in LDS, and applied to size exclusion HPLC. The elution was performed at a flow rate of 0.5 ml/min, and the absorbance (1-cm optical path length) was monitored. The protein concentration, radioactivity, and EP content in each fraction were determined.
[View Larger Version of this Image (19K GIF file)]


In a separate experiment, after quenching of phosphorylation the sample was washed four times with 4% (v/v) perchloric acid containing 20 mM Pi and 5 mM PPi in order to remove residual [gamma -32P]ATP and 32Pi. Then, the sample was solubilized and subjected to size exclusion HPLC in the same way as above. The elution profiles of proteins and radioactivities were almost the same as those in Fig. 3, but neither [gamma -32P]ATP nor 32Pi was detected (not shown). The EP contents per mg of protein in five successive fractions in the major peak of proteins were almost the same as those given in Fig. 3. These results show that EP did not decompose to an appreciable extent during the purification process.

When the SR vesicles phosphorylated with 32Pi were solubilized and subjected to size exclusion HPLC, the elution profiles of proteins and radioactivities were very similar to those in Fig. 3 (not shown). The EP content per mg of protein was again constant throughout five successive fractions in the major peak of proteins. A small amount of 32Pi was eluted at the retention time of 50 min, but it disappeared when the sample was washed twice more with the perchloric acid after quenching of phosphorylation. The EP content per mg of protein in the major peak of proteins was unaffected by these repeated washings. These results again show that EP did not decompose during the purification process.

Comparison between the Content of Phosphorylation Site and the Maximum Content of Bound FITC

The results presented so far indicate that the size exclusion HPLC provides a reliable means for purification of the FITC-labeled Ca2+-ATPase and EP. Accordingly, FITC binding and phosphorylation were determined repeatedly by this purification method. FITC labeling was performed for 240 min in which it reached the highest level (see the inset of Fig. 5). Phosphorylation was performed with [gamma -32P]ATP or 32Pi under the conditions in which almost all the phosphorylation sites are phosphorylated (18). As summarized in Table I, the content of EP was approximately half the content of bound FITC.

Table I.

The content of phosphorylation site and the maximum content of bound FITC

For the determination of FITC binding, the SR vesicles were labeled with FITC for 240 min. After the labeling was stopped by Method A, the vesicles were solubilized in LDS and applied to size exclusion HPLC. The FITC content at the peak of protein concentration was determined. The value is the mean ± S.D. for five independent experiments. For the determination of the content of the phosphorylation site, the same preparation of the SR vesicles as used for the determination of FITC binding was phosphorylated with [gamma -32P]ATP or 32Pi, solubilized in LDS, and applied to size exclusion HPLC. The EP content at the peak of protein concentration was determined. The values are the means ± S.D. for six independent experiments.
Bound FITC EP
From ATP From Pi

nmol/mg of protein
8.74  ± 0.25 4.57  ± 0.10 4.94  ± 0.13

Inhibition of FITC Labeling by Mg·ATP

In order to assess how much binding of FITC is specific to the ATP binding site, inhibition of FITC binding by Mg·ATP was investigated. The SR vesicles were labeled with FITC for 20 min in the presence of different concentrations of Mg·ATP, and the labeled Ca2+-ATPase was purified. The content of bound FITC in the purified enzyme decreased with increasing concentration of Mg·ATP and reached a constant low level at 30-60 mM Mg·ATP (Fig. 4). When the SR vesicles were labeled with FITC in the presence of 60 mM Mg·ATP, Peak 2 in reversed phase HPLC of the tryptic digest (cf. Fig. 2A) disappeared almost entirely (not shown). This indicates that labeling of Lys-515 with FITC was almost completely blocked by 60 mM Mg·ATP. On the other hand, the area of Peak 1 was unaffected by this Mg·ATP and also unaffected by extending the labeling period from 20 to 240 min in the absence and presence of 60 mM Mg·ATP. By the repeated determinations of FITC binding in the presence of 60 mM Mg·ATP, the amount of this Mg·ATP-insensitive part of bound FITC was estimated to be 0.49-0.57 nmol/mg of protein.


Fig. 4. Inhibition of FITC labeling by Mg·ATP. The SR vesicles were labeled with FITC for 20 min in the presence of different concentrations of Mg·ATP, and the labeling was stopped by Method B. The labeled vesicles were solubilized in LDS and applied to the first size exclusion HPLC. The fraction at the peak of protein concentration was applied to the second size exclusion HPLC with the same column system as in the first size exclusion HPLC. The FITC content at the peak of protein concentration in the second size exclusion HPLC was determined.
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Inhibition of ATP-induced Phosphorylation by FITC Binding

In order to examine the inhibitory effect of FITC binding on the ATP-induced phosphorylation, the SR vesicles were labeled with FITC for various periods and then phosphorylated with [gamma -32P]ATP under the conditions in which all the active phosphorylation sites remaining are phosphorylated. The Ca2+-ATPase of these vesicles was purified, and the contents of bound FITC and EP in this purified enzyme were determined (Fig. 5). FITC binding occurred rapidly in the initial 2-min period, gradually proceeded during the further incubation and then reached the highest level at 240 min (Fig. 5, inset). On the other hand, the capacity for phosphorylation decreased rapidly after the start of FITC labeling and disappeared entirely within 4 min. The relation between the content of bound FITC and that of EP formed from ATP was not linear, showing a concave curve (Fig. 5, main figure). FITC binding equivalent to about 7 nmol/mg of protein was required for complete inhibition of the ATP-induced phosphorylation. This value was appreciably higher than the total content of the phosphorylation site, but it was definitely lower than the maximum content of bound FITC (Table I).


DISCUSSION

Peptide mapping (Fig. 2A) and sequencing (see "Results") of the tryptic digest of the purified FITC-labeled Ca2+-ATPase show that Lys-515 of the enzyme is exclusively labeled with FITC. This is in agreement with the findings reported by Mitchinson et al. (12). The content of bound FITC given in Table I includes a small Mg·ATP-insensitive part of bound FITC, which is derived from Peak 1 containing no peptides. The origin of this part is obscure. By subtracting the amount of this Mg·ATP-insensitive part (0.49-0.57 nmol/mg of protein, see "Results") from the total content of bound FITC (8.74 nmol/mg of protein, Table I), the maximum content of FITC bound to Lys-515 is found to be 8.17-8.25 nmol/mg of protein. This value is in fair agreement with the theoretical value (9.06 nmol/mg of Ca2+-ATPase protein) calculated from the molecular mass (110,331 Da) of this ATPase chain (1). These findings indicate that there is one specific FITC binding site per molecule of the Ca2+-ATPase in the SR vesicles, being consistent with the previously reported results (12). Since the content of the phosphorylation site is 4.57-4.94 nmol/mg of protein (Table I), the stoichiometry of phosphorylation to FITC binding in the Ca2+-ATPase of SR vesicles is approximately 0.5:1.0. These findings lead to the conclusion that only half of the Ca2+-ATPase molecules present in the SR vesicles can be phosphorylated. This conclusion is consistent with our recent findings (8, 10) (see Introduction).

The 0.5:1.0 stoichiometry of phosphorylation to FITC binding also appears to be in harmony with the data obtained previously with SR vesicles by Mitchinson et al. (12), although they did not actually determine the content of the phosphorylation site in the SR vesicles. In those experiments Mitchinson et al. used the SR vesicles ("R1-washed") which were prepared by the method of MacLennan et al. (22) and obtained the maximum incorporation of 4.5 nmol of FITC/mg of vesicle protein. The EP level in the SR vesicles used in their experiments is expected to be as low as 1.82 nmol/mg of vesicle protein as shown by MacLennan et al. (22) (see Table II in Ref. 22).2 Therefore, it is likely that in their experiments the content of the phosphorylation site has been approximately half the maximum level of FITC binding.

It is improbable that the observed lack of phosphorylation in half of the Ca2+-ATPase molecules is due to a possible denaturation, because FITC shows a highly specific reactivity toward Lys-515 in almost all the Ca2+-ATPase molecules and because FITC labeling of Lys-515 in almost all the Ca2+-ATPase molecules is blocked by Mg·ATP (Fig. 4 and see "Results"). This conclusion is supported by the earlier findings to show that an inactive portion of the Ca2+-ATPase in preparations of SR vesicles does not bind FITC (15) and that the high specific reactivity of Lys-515 toward FITC requires special steric conditions only given in the native conformation of the active site (23, 24). Intactness of all the Ca2+-ATPase molecules present in the SR vesicles used is further supported by our previous observations (8) that all the Ca2+-ATPase molecules in the SR vesicles are equally available for tight binding of Mg2+ and F- and that this tight binding is entirely prevented by Ca2+ binding to the high-affinity Ca2+ sites. The lack of phosphorylation in half of the Ca2+-ATPase molecules may be possibly due to an intermolecular ATPase-ATPase interaction in the SR vesicles.

FITC binding completely inhibits phosphorylation before the binding reaches its maximum level (Fig. 5). This finding indicates that FITC preferentially binds to a part (possibly half) of the Ca2+-ATPase molecules in the SR vesicles and that this binding is primarily responsible for the inhibition of phosphorylation. The above preferential binding is again likely due to an intermolecular ATPase-ATPase interaction. It seems possible that the intermolecular ATPase-ATPase interaction is partially disrupted by detergents, because it was reported previously (12, 14, 15) that 0.89-0.97 mol of bound FITC/mol of Ca2+-ATPase molecule is required for complete inhibition of the detergent-purified Ca2+-ATPase.

Recently, Liu et al. (25) have presented the data suggesting that half of the alpha -subunits of membrane-bound Na+,K+-ATPase are dormant (i.e. the half cannot be phosphorylated). It is possible that the half-of-the sites reactivity in the membrane-bound Na+,K+-ATPase corresponds to that indicated with the Ca2+-ATPase of SR vesicles in the present study.


FOOTNOTES

*   This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture, Japan.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be 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; E-mail: kanazawa{at}asahikawa-med.ac.jp.
1   The abbreviations used are: SR, sarcoplasmic reticulum; EP, phosphoenzyme; FITC, fluorescein 5-isothiocyanate (isomer I); MOPS, 3-(N-morpholino)propanesulfonic acid; MES, 2-(N-morpholino)ethanesulfonic acid; LDS, lithium dodecyl sulfate; HPLC, high performance liquid chromatography.
2   The EP level obtained under the conditions of MacLennan et al. (22) (0.13 mM [gamma -32P]ATP, 0.1 mM CaCl2, 0.1 mM EGTA, 5 mM MgCl2, 0.1 M KCl (pH 6.8), 0 °C) is 96% of the maximum EP level obtained with [gamma -32P]ATP under the conditions of Barrabin et al. (18) which are described under "Experimental Procedures" (H. Suzuki, and T. Kanazawa, unpublished observations).

Acknowledgments

S. N. is grateful to Prof. Hajime Iizuka (Department of Dermatology, Asahikawa Medical College) for his continued encouragement during this work.


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