(Received for publication, April 28, 1997)
From the Department of Biochemistry, Asahikawa Medical College, Nishikagura Asahikawa 078, Japan
Sarcoplasmic reticulum vesicles were modified
with 1,2-cyclohexanedione (CHD), a specific arginine-modifying reagent,
in sodium borate (pH 8.0 or 8.8). Phosphoenzyme formation from
Pi in the Ca2+-ATPase (reversal of
hydrolysis of the phosphoenzyme intermediate) was almost completely
inhibited by the modification with CHD. Tight binding of
F and Mg2+ and high affinity binding of
vanadate in the presence of Mg2+, either of which produces
a transition state analog for phosphoenzyme formation from the
magnesium-enzyme-phosphate complex, were also markedly inhibited. In
contrast, phosphoenzyme formation from acetyl phosphate in the forward
reaction was unaffected. The enzyme was appreciably protected by tight
binding of F
and Mg2+ or by high affinity
binding of vanadate in the presence of Mg2+, but not by the
presence of 20 mM MgCl2 alone or 150 mM Pi alone, against the CHD-induced inhibition
of phosphoenzyme formation from Pi. Peptide mapping of the
tryptic digests, detection of peptides containing CHD-modified arginyl
residues with Girard's reagent T, sequencing, and mass spectrometry
showed that Arg-198 was a single major residue protected by tight
binding of F
and Mg2+ against the
modification with CHD. These results indicate that modification of
Arg-198 with CHD is responsible for at least a part (the portion
reduced by the transition state analogs) of the CHD-induced inhibition
of phosphoenzyme formation from Pi and suggest that Arg-198
is located in or close to the catalytic site in the transition state
for phosphoenzyme formation from the magnesium-enzyme-phosphate
complex.
The SR1
Ca2+-ATPase is a 110-kDa membrane-bound protein, the
primary structure of which has been revealed (1). This enzyme catalyzes
Ca2+ transport coupled to ATP hydrolysis (2, 3). The enzyme is activated by Ca2+ binding to the high affinity
Ca2+ binding sites, and then the -phosphoryl group of
Mg·ATP bound to the ATP binding site is transferred to Asp-351 (1,
4-6) to form an EP intermediate (7, 8). A subsequent
conformational change of the EP results in Ca2+
release to the lumen (9). Finally, the EP is hydrolyzed to form Pi and the dephosphoenzyme. Acetyl phosphate also
serves as a substrate through EP formation (10, 11). The
EP can be formed from Pi in the presence of
Mg2+ and absence of Ca2+ by reversal of the
late step of the catalytic cycle (12, 13). This EP formation
occurs through a magnesium-enzyme-phosphate complex that is
formed by random binding of Mg2+ and Pi to the
enzyme (14, 15).
CHD is a chemical modification reagent that reacts specifically with the guanidino group of arginyl residues to produce a stable product, DHCH-Arg (16). Recently, we have shown that binding of Mg·ATP or Mg·ADP to the ATP binding site of the Ca2+-ATPase is completely inhibited by the modification with CHD and that Arg-489 and Arg-678 are involved in this inhibition (17). The findings have led to the conclusion that these arginyl residues are located at the ATP binding site. We have further shown that EP formation from Pi has also been inhibited by the modification with CHD and suggested that the arginyl residue(s) involved in this inhibition is distinct from the above residues.
It was previously shown (18-20) that F and
Mg2+ bind simultaneously and tightly to the catalytic site
of this enzyme to form a stable transition state analog for
EP formation from the magnesium-enzyme-phosphate complex.
Vanadate also binds with high affinity to the enzyme in the presence of
Mg2+ to form a transition state analog for this
EP formation (21, 22).
In the present study, to identify the arginyl residue(s) involved in
the CHD-induced inhibition of EP formation from
Pi, we have modified the SR Ca2+-ATPase with
CHD and examined effects of the above transition state analogs on this
modification. We have found that the enzyme has been appreciably
protected by the transition state analogs against the CHD-induced
inhibition of EP formation from Pi. Peptide mapping of tryptic digests of the CHD-modified enzyme, sequencing, and
mass spectrometry have shown that Arg-198 is a single major residue
protected by tight binding of F and Mg2+
against the modification with CHD. The results indicate that modification of Arg-198 with CHD is responsible for at least a part
(the portion reduced by the transition state analogs) of the inhibition
of EP formation from Pi and suggest that Arg-198 is located in or close to the catalytic site in the transition state
for EP formation from the magnesium-enzyme-phosphate
complex.
SR vesicles were prepared from
rabbit skeletal muscle and stored at 80 °C as described previously
(23). The content of phosphorylation site determined with
[
-32P]ATP according to Barrabin et al. (24)
was 4.00 ± 0.06 nmol/mg (n = 6).
Pretreatment of the SR vesicles with
F and Mg2+ was performed as described
previously (20) with slight modifications. The vesicles (2 mg/ml) were
incubated at 25 °C for 3 h in 1 mM KF, 10 mM MgCl2, 1 mM EGTA, 20% (v/v)
Me2SO, 20% (v/v) glycerol, 0.1 M KCl, and 40 mM imidazole/HCl (pH 7.5), unless otherwise stated. The
reaction was quenched by diluting the mixture twice with an ice-cold
solution containing 0.1 mM CaCl2, 0.1 M KCl, 0.3 M sucrose, and 5 mM
MOPS/Tris (pH 7.0). The resulting vesicles were washed by
centrifugation once with this solution.
Modification was started at 37 °C by adding CHD (dissolved in 50 mM sodium borate (pH 8.0 or 8.8)) to a suspension of the SR vesicles to give a final composition as described in the figure legends. The modification was quenched at 0 °C by one of the following methods. In Method I, the mixture was applied to a centrifuge column (10-ml disposable syringe filled with coarse Sephadex G-50) preequilibrated with a solution (Solution A) containing 0.1 mM CaCl2, 0.1 M KCl, 20 mM MOPS, and 5 mM sodium borate (pH 7.0). In Method II, the mixture was centrifuged, and the pellet was washed with Solution A and suspended in this solution. In Method III, the mixture was centrifuged, and the pellet was washed with 50 mM sodium borate (pH 8.0) and suspended in this buffer.
Determination of Tightly Bound FThe SR vesicles were treated with
F and Mg2+ as described above. In the control
samples, CaCl2 was added to the incubation medium to give
0.1 mM free Ca2+ under the otherwise same
conditions as above (tight binding of F
and
Mg2+ is prevented by 0.1 mM Ca2+
(20)). The treated vesicles were washed by centrifugation four times
with a solution containing 1 µM A23187, 2 mM
EDTA, 10% (v/v) Me2SO, 0.1 M KCl, and 5 mM MOPS/NaOH (pH 7.0) and suspended in deionized water.
Magnesium bound to the vesicles was extracted with 0.8 N
HNO3, and the concentration of magnesium in the extract was
determined by atomic absorption spectrophotometry as described previously (19). The content of tightly bound Mg2+ was
obtained by subtracting the content of magnesium in the extract from
the control sample. Fluoride bound to the vesicles was extracted by
incubating the vesicles at 95 °C for 5 min in 5 mM
HEPES/KOH (pH 8.0). The sample was then centrifuged to remove insoluble materials, and KNO3 was added to the supernatant to give
0.1 M. The sample was adjusted to pH 3.0 with citric acid,
and the concentration of F
was measured by use of a
fluoride-selective electrode as described previously (19). The content
of tightly bound F
was obtained by subtracting the
content of F
in the extract from the control sample.
The SR vesicles (0.2 mg/ml)
were incubated at 25 °C for 30 min in various concentrations of
vanadate, 40% (v/v) Me2SO, 0.1 M KCl, 30 mM MOPS, 7 mM sodium borate (pH 7.0), and
others as described in the legend to Fig. 3. The mixture was
centrifuged, and the pellet was dissolved in 2% (w/v) SDS and 0.1 N NaOH. The solution was neutralized with HCl, and the
concentration of vanadate was measured by the method of Goodno
(25).
Proteolysis, Peptide Mapping, Detection of Peptides Containing DHCH-Arg, Sequencing, and Mass Spectrometry
The CHD-modified SR vesicles (1 mg/ml) were digested with TPCK-treated trypsin (0.2 mg/ml) at 40 °C for 4 h in 10 mM CaCl2 and 50 mM sodium borate (pH 8.0). After centrifugation, the supernatant was subjected to reversed phase HPLC that was performed at a flow rate of 1 ml/min as described previously (26). The absorbance of peptides was monitored at 214 nm, and fractions of 0.3-1.4 ml each were collected. Peptides containing DHCH-Arg in the fractions were detected at 325 nm by the method of Patthy et al. (27) using Girard's reagent T. It was difficult to determine the content of DHCH-Arg in the CHD-modified vesicles by this method because the background level of the absorbance was too high even after solubilization of the vesicles with SDS. Sequencing of isolated peptides was performed with an Applied Biosystems 477A/120A sequencer. For mass spectrometry, the isolated peptides were dissolved in 0.1% trifluoroacetic acid and 0.3% 1-thioglycerol and infused into the frit-FAB (fast atom bombardment) probe of a Jeol JMS-SX102 spectrometer at a flow rate of 6 µl/min by use of a microprocessor-controlled Pump 22 infusion syringe pump (Harvard Apparatus, Inc., South Natick, MA). The mobile phase composition was 0.1% trifluoroacetic acid, 30% methanol, and 0.3% 1-thioglycerol in water (v/v). The accelerating voltage was 10 kV, and ions were analyzed in the positive mode as a function of their m/z ratio.
Phosphorylation of Ca2+-ATPasePhosphorylation of the SR vesicles (0.4 mg/ml) with 1 mM 32Pi was performed at 25 °C for 10 min in 20 mM MgCl2, 5 mM EGTA, 40% (v/v) Me2SO, 30 mM MOPS, and 7 mM sodium borate (pH 7.0). The reaction was quenched with trichloroacetic acid containing Pi. Phosphorylation of the vesicles (1 mg/ml) with 3 mM acetyl [32P]phosphate was performed at 25 °C in 5 mM MgCl2, 2 mM CaCl2, 50 mM KCl, 50 mM Tris, and 20 mM sodium borate (pH 8.0). The reaction was quenched with trichloroacetic acid containing nonradioactive acetyl phosphate. The amount of EP formed was determined as described previously (19).
Miscellaneous MethodsCHD was purchased from Aldrich.
Girard's reagent T and KF were from Nacalai Tesque (Kyoto, Japan). ATP
was from Yamasa Biochemicals (Choshi, Japan). Acetyl phosphate was from
Kohjin (Tokyo, Japan). TPCK-treated trypsin and
Na3VO4 were from Sigma.
[-32P]ATP was obtained from NEN Life Science Products.
Vanadate solutions were prepared from Na3VO4
according to Goodno (25) just before use. 32Pi
was purified according to Kanazawa and Boyer (12). Acetyl [32P]phosphate was prepared by Procedure B in the method
of Stadtman (28). Protein concentrations were determined by the method
of Lowry et al. (29) with bovine serum albumin as a
standard. Data were analyzed by the nonlinear least squares method
according to the algorithm of Marquardt (30) as described previously
(19).
As shown in Fig.
1A, when the SR vesicles were
pretreated with neither F nor Mg2+ and then
treated with CHD, EP formation from Pi was
inhibited with pseudo-first order kinetics and fell to 13% of the
original level in 90 min (
). When the vesicles were pretreated with
F
and Mg2+ and then treated with CHD, the
enzyme was protected appreciably against the CHD-induced inhibition of
EP formation (tightly bound F
and
Mg2+ were removed before the determination of EP
formation) (
). The pretreatment without F
(
) or
Mg2+ (
) provided no protection. The observed first order
kinetics of the CHD-induced inhibition of EP formation (Fig.
1, A-C, solid lines) is consistent with the
possibility that this inhibition was caused by modification of a single
residue. However, it is also possible that more than one vital arginyl
residue was modified at an approximately equal rate.
CHD-induced inhibition of EP
formation from Pi and protection by pretreatment with
F and Mg2+, by the presence of vanadate and
Mg2+, or by EP formation from Pi
against the inhibition. A, the SR vesicles were pretreated
with KF and MgCl2 (
,
), without KF and with
MgCl2 (
,
), with KF and without MgCl2 in
the presence of 5 mM EDTA and absence of EGTA (
,
),
or with neither KF nor MgCl2 in the presence of 5 mM EDTA and absence of EGTA (
,
), otherwise as
described under "Experimental Procedures." The vesicles (2 mg/ml)
were then incubated with (
,
,
,
) or without (
,
,
,
) 4 mM CHD for various times in 2 mM
EDTA, 20% (v/v) Me2SO, and 30 mM sodium borate
(pH 8.8). The reaction was quenched by Method I. The vesicles were
further incubated at 25 °C for 60 min in 20 mM
CaCl2, 0.1 M KCl, 20 mM MOPS, and 5 mM sodium borate (pH 7.0) (tightly bound F
and Mg2+ are entirely released by incubation with 20 mM Ca2+ (19)) and then passed through a
centrifuge column preequilibrated with a solution (Solution B) containing 40 mM
MgCl2, 10 mM EGTA, 20 mM MOPS, and
5 mM sodium borate (pH 7.0). EP formation from 32Pi was determined. Solid and
dashed lines show least squares fit to a single exponential
in which the first order rate constants were 0.024 and 0.012 min
1, respectively. B, the SR vesicles (2.1 mg/ml) were preincubated at 37 °C for 30 min in 1.04 mM
EGTA, 20.8% (v/v) Me2SO, and 31.3 mM sodium
borate (pH 8.8) in the presence of 0.52 mM vanadate and
20.8 mM MgCl2 (
,
), in the absence of
vanadate and presence of 20.8 mM MgCl2 (
,
), in the presence of 0.52 mM vanadate and 2.1 mM EDTA and absence of MgCl2 and EGTA (
,
), or in the absence of vanadate, MgCl2, and EGTA and
presence of 2.1 mM EDTA (
,
). After the addition of
sodium borate (pH 8.8) with (
,
,
,
) or without (
,
,
,
) CHD, the vesicles were further incubated for various times.
The final composition was 2 mg of the vesicles/ml, 0 or 4 mM CHD, 0 or 0.5 mM vanadate, 0 or 20 mM MgCl2, 0 or 2 mM EDTA, 0 or 1 mM EGTA, 20% (v/v) Me2SO, and 30 mM sodium borate (pH 8.8). The reaction was quenched by
Method I. The vesicles were then incubated with 3 mM
CaCl2 and 2 mM ATP at 25 °C for 30 min in
0.1 M KCl, 20 mM MOPS, and 5 mM
sodium borate (pH 7.0) (bound vanadate and Mg2+ are
entirely released by this incubation (45 and 46)). The vesicles were
passed through two successive centrifuge columns, the first column
being preequilibrated with Solution A and the second column with
Solution B. EP formation from 32Pi
was determined. Solid and dashed lines show least
squares fit to a single exponential in which the first order rate
constants were 0.023 and 0.011 min
1, respectively.
C, the SR vesicles (2.1 mg/ml) were preincubated in the
presence of 1.04 mM Pi and 8.33 mM
MgCl2 at 37 °C for 15 min in 2.08 mM EGTA,
31.3% (v/v) Me2SO, and 31.3 mM sodium borate (pH 8.0) (
,
). The amount of EP formed from
Pi by this preincubation was 4.1 nmol/mg. In other
experiments, the vesicles were preincubated in the absence of
Pi and presence of 8.33 mM MgCl2
(
,
), in the presence of 156 mM Pi and
2.1 mM EDTA and absence of MgCl2 and EGTA (
,
), or in the absence of Pi, MgCl2, and EGTA
and presence of 2.1 mM EDTA (
,
), as above (no
EP is formed under these conditions). After the addition of
sodium borate (pH 8.0) with (
,
,
,
) or without (
,
,
,
) CHD, the vesicles were further incubated for various times.
The final composition was 2 mg of the vesicles/ml, 0 or 15 mM CHD, 0, 1, or 150 mM Pi, 0 or 8 mM MgCl2, 0 or 2 mM EDTA, 0 or 2 mM EGTA, 30% (v/v) Me2SO, and 30 mM sodium borate (pH 8.0). The reaction was quenched by Method II. The vesicles were then passed through a centrifuge column
preequilibrated with Solution B. EP formation from
32Pi was determined. Solid and
dashed lines show least squares fit to a single exponential,
in which the first order rate constants were 0.031 and 0.018 min
1, respectively.
Protection by Presence of Vanadate and Mg2+ against CHD-induced Inhibition of EP Formation from Pi
As
shown in Fig. 1B, when the SR vesicles were treated with CHD
in the presence of both vanadate and Mg2+, the enzyme was
protected against the CHD-induced inhibition of EP formation
from Pi (bound vanadate and Mg2+ were removed
before the determination of EP formation) (). In the
absence of vanadate and presence of 20 mM Mg2+
(
) or in the presence of 0.5 mM vanadate and absence of
Mg2+ (
), no protection was observed.
As shown in Fig.
1C, when the SR vesicles were preincubated with
Pi in the presence of Mg2+ and absence of
Ca2+ (4.1 nmol of EP/mg of SR vesicles was
formed by this preincubation) and then treated with CHD, the enzyme was
protected against the CHD-induced inhibition of EP formation
from Pi (). However, this protection was somewhat less
effective than the protection by the pretreatment with F
and Mg2+ or by the presence of vanadate and
Mg2+ (compare Fig. 1C with Fig. 1, A
and B). Binding of Pi to the catalytic site in
the presence of 150 mM Pi and absence of
Mg2+ provided no protection (
).
The SR vesicles were treated with or without CHD
for various times, and then tight binding of F and
Mg2+ was determined (Fig. 2).
The amounts of tightly bound F
and Mg2+ at
zero time of the treatment were 14.7 and 8.4 nmol/mg, respectively, which are 3.7 and 2.1 times the content of phosphorylation site, respectively (4.0 nmol/mg, see "Experimental Procedures"). This is
in agreement with our previous conclusion (20) that the stoichiometry of tight binding of F
and Mg2+ to the maximum
level of phosphorylation is 4:2:1. The tight binding was inhibited
progressively during the treatment with CHD, although the inhibition
was considerably slower than the CHD-induced inhibition of
EP formation from Pi. This less effective
inhibition may be in part due to the fact (18, 19) that the binding of
F
and Mg2+ to the enzyme is virtually
irreversible in the absence of Ca2+.
CHD-induced Inhibition of Vanadate Binding and Protection by Pretreatment with F
The SR vesicles were pretreated with or without
F in the presence of Mg2+ and then treated
with or without CHD for 90 min. After tightly bound F
and
Mg2+ were removed, vanadate binding was determined at
various concentrations of vanadate in the presence and absence of
Mg2+ (Fig. 3A). In
the control (
,
), in which the vesicles were pretreated without
F
and then treated without CHD,
Mg2+-dependent vanadate binding (broken
line) increased with increasing concentrations of vanadate and was
saturated with 10 µM vanadate. The maximum level of this
binding was in substantial agreement with the content of the
phosphorylation site. The Mg2+-dependent
binding was almost completely inhibited by the treatment with CHD (
,
). When the vesicles were pretreated with F
and
Mg2+ and then treated with CHD, the enzyme was appreciably
protected by this pretreatment against the CHD-induced inhibition of
Mg2+-dependent vanadate binding (
,
).
When the vesicles were pretreated without F
and then
treated with CHD for various times, vanadate binding was inhibited with
pseudo-first order kinetics at a rate of 0.028 min
1 (Fig.
3B, line drawn through open squares).
This rate of inhibition was in good agreement with the rate
(0.023-0.024 min
1) of the CHD-induced inhibition of
EP formation from Pi under the same conditions
(see solid lines in Fig. 1, A and B).
The inhibition of vanadate binding was slowed by the pretreatment with
F
and Mg2+ (Fig. 3B,
line drawn through open circles) to a similar
extent as the inhibition of EP formation from Pi
(cf. Fig. 1A).
The SR vesicles were pretreated with or without
F in the presence of Mg2+. In the experiments
shown in Fig. 4A, the
pretreated vesicles were treated with or without CHD for various times.
After tightly bound F
and Mg2+ were removed,
EP formation from acetyl phosphate was determined. CHD
caused no substantial inhibition of the EP formation when the vesicles were pretreated without F
(compare
with
). The pretreatment with F
and Mg2+
exerted a slight protective effect during the control incubation without CHD (compare
with
). In the experiments shown in Fig. 4B, the pretreated vesicles were treated with or without CHD
for 90 min and then phosphorylated with acetyl phosphate for various times. When the vesicles were pretreated without F
and
treated with CHD, the rate of EP formation was essentially the same as that of EP formation in native SR vesicles
(compare
with ×). The pretreatment with F
and
Mg2+ had virtually no effect on the kinetics of
EP formation in the CHD-treated vesicles (compare
with
). When the vesicles were pretreated without F
and
treated without CHD, EP formation was appreciably faster than that in the native vesicles for unknown reasons (compare
with
×).
Peptide Mapping of Tryptic Digests of CHD-modified SR Vesicles
In the first series of experiments (Fig.
5, A-D), the SR vesicles were
pretreated with neither F nor Mg2+
(A), with F
and without Mg2+
(B), without F
and with Mg2+
(C), or with F
and Mg2+
(D) and then treated with CHD. After tightly bound
F
and Mg2+ were removed, the vesicles were
digested with TPCK-treated trypsin and subjected to reversed phase
HPLC. The peptide maps at 214 nm of the digests (Fig. 5,
A-D, lower traces) agreed closely with each
other. The absorbance at 325 nm of peptides containing DHCH-Arg in the
peaks indicated by arrows were strongly reduced by the pretreatment with F
and Mg2+ (compare
upper trace of Fig. 5D with upper
trace of Fig. 5A) but not by the pretreatment without
F
or Mg2+ (compare upper traces of
Fig. 5, B and C, with upper trace of Fig. 5A).
Peptide mapping of tryptic digests of
CHD-modified SR vesicles and effects of pretreatment with
F and Mg2+, of the presence of vanadate and
Mg2+, or of EP formation from Pi on
the modification with CHD. In the first series of experiments
(A-D), the SR vesicles were pretreated with neither KF nor MgCl2
in the presence of 5 mM EDTA and absence of EGTA
(A), with KF and without MgCl2 in the presence of 5 mM EDTA and absence of EGTA (B), without KF
and with MgCl2 (C), or with KF and
MgCl2 (D), otherwise as described under
"Experimental Procedures." The vesicles were then incubated with 4 mM CHD for 45 min in 2 mM EDTA, 20% (v/v)
Me2SO, and 30 mM sodium borate (pH 8.8). The
reaction was quenched by Method III. The vesicles were further
incubated with 20 mM CaCl2 in 50 mM
sodium borate (pH 8.0) at 25 °C for 60 min (to remove tightly bound
F
and Mg2+). In the second series of
experiments (E-G), the SR vesicles were preincubated in the
absence of vanadate and presence of MgCl2 (E),
in the presence of vanadate and 2.1 mM EDTA and absence of MgCl2 and EGTA (F), or in the presence of
vanadate and MgCl2 (G), as in Fig.
1B. The vesicles were then incubated with 4 mM
CHD for 45 min in 0 or 0.5 mM vanadate, 0 or 20 mM MgCl2, 0 or 2 mM EDTA, 0 or 1 mM EGTA, 20% (v/v) Me2SO, and 30 mM sodium borate (pH 8.8). The reaction was quenched by
Method III. The vesicles were further incubated with 3 mM
CaCl2 and 2 mM ATP in 50 mM sodium borate (pH 8.0) at 25 °C for 30 min (to remove bound vanadate and
Mg2+). In the third series of experiments
(H-J), the SR vesicles were preincubated in the absence of
Pi and presence of 8.33 mM MgCl2 (H), in the presence of 156 mM Pi
and 2.1 mM EDTA and absence of MgCl2 and EGTA
(I), or in the presence of 1.04 mM
Pi and 8.33 mM MgCl2
(J), as in Fig. 1C. The vesicles were then
incubated with 15 mM CHD for 45 min in 0, 1, or 150 mM Pi, 0 or 8 mM MgCl2, 0 or 2 mM EDTA, 0 or 2 mM EGTA, 30% (v/v)
Me2SO, and 30 mM sodium borate (pH 8.0). The
reaction was quenched by Method III. In A-J, the vesicles
were digested with TPCK-treated trypsin and subjected to reversed phase
HPLC. Elution was performed with the following linear gradient of
acetonitrile in 0.1% trifluoroacetic acid: 0% from 0 to 20 min, 10%
at 40 min, 20% at 80 min, 35% at 118 min, 50% at 128 min, and 100%
at 138 min. Fractions of 0.3 ml each were collected. The absorbance at
214 nm (lower traces) and the absorbance of peptides
containing DHCH-Arg at 325 nm (upper traces) were
monitored.
In the second series of experiments (Fig. 5, E-G), the SR vesicles were treated with CHD in the absence of vanadate and presence of Mg2+ (E), in the presence of vanadate and absence of Mg2+ (F), or in the presence of vanadate and Mg2+ (G). After bound vanadate and Mg2+ were removed, the vesicles were digested with TPCK-treated trypsin and subjected to reversed phase HPLC. The peaks indicated by arrows were substantially reduced only when both vanadate and Mg2+ were present (compare upper trace of Fig. 5G with upper traces of Fig. 5, E and F).
In the third series of experiments (Fig. 5, H-J), the SR vesicles were preincubated in the absence of Pi and presence of 8.33 mM MgCl2 (H), in the presence of 156 mM Pi and absence of Mg2+ (I), or in the presence of 1.04 mM Pi and 8.33 mM MgCl2 (J) and then treated with CHD. The vesicles were digested with TPCK-treated trypsin and subjected to reversed phase HPLC. The preincubation in the presence of Pi and Mg2+ (4.1 nmol of EP per mg of SR vesicles was formed by this preincubation) induced a small but definite reduction in the peaks indicated by arrows (compare upper trace of Fig. 5J with upper trace of Fig. 5H). Binding of Pi to the catalytic site in the presence of 150 mM Pi and absence of Mg2+ had no effect on these peaks (compare upper trace of Fig. 5I with upper trace of Fig. 5H).
It is clear from the chromatographic profiles that the peaks reduced by
the presence of vanadate and Mg2+ (Fig. 5, E-G,
arrows) and by the presence of Pi and
Mg2+ (Fig. 5, H-J, arrows)
corresponded to those reduced by the pretreatment with F
and Mg2+ (Fig. 5, A-D, arrows).
The SR vesicles
were pretreated without F and with Mg2+ (Fig.
6A) or with F
and Mg2+ (Fig. 6B) and then treated with CHD.
After tightly bound F
and Mg2+ were removed,
the vesicles were digested with TPCK-treated trypsin and subjected to
the first reversed phase HPLC (Fig. 6, A and B).
Fraction I and fraction II in the figures were pooled separately and subjected to the second reversed phase HPLC (Fig. 6, C--F). Fraction I in Fig. 6A gave a single major peak at 325 nm (upper trace of Fig. 6C, fraction I). Fraction I in Fig. 6B gave a major peak at 325 nm at the same retention time as in Fig. 6C (upper trace of Fig. 6D, fraction I), but this peak was much smaller than the corresponding peak in Fig. 6C. Fraction II in Fig. 6A gave two close peaks at 325 nm (upper trace of Fig. 6E, fraction II). These two peaks were greatly reduced (upper trace of Fig. 6F, fraction II) when HPLC was performed with fraction II in Fig. 6B.
Peptides containing DHCH-Arg in fraction I in Fig. 6 (C and D) and fraction II in Fig. 6 (E and F) were further purified by the third reversed phase HPLC (Fig. 6, G-J). Fraction I in Fig. 6C gave a major peak and a minor peak at 325 nm (upper trace of Fig. 6G, peak Ia and peak Ib). Fraction I in Fig. 6D also gave a major peak and a minor peak at 325 nm (upper trace of Fig. 6H, peak Ia and peak Ib), but these peaks were much smaller than the corresponding peaks in Fig. 6G. Fraction II in Fig. 6E gave two major peaks at 325 nm (upper trace of Fig. 6I, peak IIa and peak IIb). Fraction II in Fig. 6F also gave two major peaks at 325 nm (upper trace of Fig. 6J, peak IIa and peak IIb), but these two peaks were much smaller than the corresponding peaks in Fig. 6I.
Sequencing and Mass Analysis of Peptides Containing DHCH-ArgThe peptides containing DHCH-Arg isolated as above (peak
Ia and peak Ib in Fig. 6, G and
H, and peak IIa and peak IIb in Fig. 6, I and J) were sequenced (Table
I). All the isolated peptides gave the
same sequence (His-190 to Lys-204) in the Ca2+-ATPase, in
which a missing residue (X) corresponded to Arg-198. These
results suggest that Arg-198 in the Ca2+-ATPase was
modified with CHD. This is consistent with the previously reported
findings (16) that carboxyl-terminal peptide bonds of CHD-modified
arginyl residues are resistant to tryptic cleavage. The molecular
masses of these peptides determined by mass spectrometry were in good
agreement with the monoisotopic mass calculated from the above sequence
on the assumption that X is DHCH-Arg (Table II). These findings show that Arg-198 in
the Ca2+-ATPase was modified with CHD and that this
modification of Arg-198 was strongly inhibited by the pretreatment with
F and Mg2+. Furthermore, it is very likely
that the modification of Arg-198 with CHD was specifically inhibited
also by the presence of vanadate and Mg2+ (Fig. 5,
E-G, arrows) or by the presence of
Pi and Mg2+ (Fig. 5, H-J,
arrows). The reason why peptides with the same sequence and
same modification gave different retention times in HPLC remains
obscure.
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The observed protection by tight binding of F and
Mg2+ (Fig. 1A) or by high affinity binding of
vanadate (Fig. 1B) against the CHD-induced inhibition of
EP formation from Pi suggests that an arginyl
residue(s) protected by these transition state analogs contributes
toward formation of the transition state for EP formation from the magnesium-enzyme-phosphate complex or, alternatively, that the
protected arginyl residue(s) is located close to the essential
components (bound phosphate, bound Mg2+, or other
functional groups) of this transition state. This view is consistent
with the findings that binding of the transition state analogs is
inhibited by the modification with CHD (Figs. 2 and 3) and that the
enzyme is protected by tight binding of F
and
Mg2+ against the CHD-induced inhibition of high affinity
vanadate binding (Fig. 3). It is also in harmony with the observed lack of protection by the enzyme-magnesium (Fig. 1B) and
enzyme-phosphate (Fig. 1C) complexes, because the structures
of these complexes should be different from the structure of the
transition state for EP formation from the
magnesium-enzyme-phosphate complex.
The reduction in two specific peaks by tight binding of F
and Mg2+ (Fig. 5D, arrows) or by high
affinity binding of vanadate (Fig. 5G, arrows)
indicates that a few specific arginyl residues are selectively
protected by the transition state analogs against the modification with
CHD. The data from peptide purification (Fig. 6), sequencing (Table I),
and mass spectrometry (Table II) show that the only major arginyl
residue protected by these analogs is Arg-198. These findings indicate
that modification of Arg-198 with CHD is responsible for at least a
part (the portion reduced by the transition state analogs) of the
CHD-induced inhibition of EP formation from Pi
(Fig. 1, A and B). Dux and Martonosi (31) showed
previously that tryptic cleavage of the Ca2+-ATPase at
Arg-198 is inhibited by high affinity binding of vanadate to the
enzyme. Their findings appear to be compatible with our present
observations.
The lack of protection by the presence of 20 mM Mg2+ alone against the CHD-induced inhibition of EP formation from Pi (Fig. 1B) and against the modification of Arg-198 with CHD (Fig. 5E, arrows) indicates that Arg-198 is not located at the Mg2+ binding site in the enzyme-magnesium complex. In addition, the lack of protection by the presence of 150 mM Pi alone against the CHD-induced inhibition of EP formation from Pi (Fig. 1C) and against the modification of Arg-198 with CHD (Fig. 5I, arrows) indicates that Arg-198 is not located at the Pi binding site in the enzyme-phosphate complex. The slightly less effective protection by EP formation from Pi against the CHD-induced inhibition of EP formation from Pi (Fig. 1C) and against the modification of Arg-198 with CHD (Fig. 5J, arrows) suggests that Arg-198 is located close to, but outside, the phosphorylation site. The location of Arg-198 in proximity to the phosphorylation site is consistent with the previously reported findings (32) that tryptic cleavage of the Ca2+-ATPase at Arg-198 is inhibited by EP formation from Pi.
No appreciable effects of the modification with CHD on the extent (Fig. 4A) and rate (Fig. 4B) of EP formation from acetyl phosphate indicate that phosphoryl transfer from the bound substrate to the phosphorylation site in the forward reaction is not substantially affected by the modification of Arg-198. This finding also shows definitely that Arg-198 is located outside the phosphorylation site.
Chen et al. (33) showed previously that binding of
,
-bidentate CrATP to the catalytic site of the
Ca2+-ATPase is inhibited by Pi or vanadate in
the presence of Mg2+, that the chromium moiety of the bound
CrATP accepts a ligand from the enzyme to form an exchange inert
coordination complex, and that the site of CrATP attachment through
this stable coordinate bond is situated on the A2 (32)
tryptic fragment (Ac-Met-1 to Arg-198). Their observations are
consistent with our present view that Arg-198 is located in or close to
the catalytic site in the transition state for EP formation
from magnesium-enzyme-phosphate complex.
Arg-198 is conserved in sarco(endo)plasmic reticulum Ca2+-ATPases (1, 34-36) but not in other P-type ATPases including the Na+,K+-ATPase (37, 38), H+,K+-ATPase (39, 40), and plasma membrane Ca2+-ATPase (41). Therefore, although it is possible that Arg-198 is directly involved in the catalytic process of EP formation from Pi, it appears more likely that the observed inhibition (at least the portion reduced by the transition state analogs) is due to steric hindrance induced by CHD-modified Arg-198.
The secondary structural model for the Ca2+-ATPase suggests
that the enzyme is composed of 10 transmembrane -helices
(M1-M10) and a cytoplasmic globular fraction
that is divided into two main domains, a small cytoplasmic loop
(Ala-132-Asp-237 between M2 and M3) and a
large cytoplasmic loop (Asn-330-Phe-740 between M4 and
M5) (1). The large cytoplasmic loop contains the
phosphorylation site and the ATP binding site. The functional role of
the small cytoplasmic loop is less clear. However, in the previous
studies on the function of this region, it was shown that the
conformational change of EP is blocked by site-directed
mutations of Thr-181, Gly-182, Glu-183 (42), and Gly-233 (43) and by a
tryptic cleavage at Lys-234 or Arg-236 (44). It was also shown (43)
that the affinity for Pi in EP formation from
Pi is reduced in the Gly-233 mutants. Accordingly, it is
likely that this small cytoplasmic loop including Arg-198 also
contributes in part to the catalytic site.
We thank Professor Akimasa Okuno (Department of Pediatrics, Asahikawa Medical College) for his continued encouragement during this work.