Modification of Histidine 5 in Sarcoplasmic Reticulum Ca2+-ATPase by Diethyl Pyrocarbonate Causes Strong Inhibition of Formation of the Phosphoenzyme Intermediate from Inorganic Phosphate*

Sarcoplasmic reticulum vesicles were modified with diethyl pyrocarbonate (DEPC), a histidine-modifying reagent. Phosphoenzyme formation from Pi in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by this modification. Tight binding of F− and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, both of which produce transition state analogs for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also inhibited. Formation of the phosphoenzyme from acetyl phosphate in the forward reaction was only weakly inhibited, but hydrolysis of the phosphoenzyme was strongly inhibited. The enzyme was protected by tight binding of F−and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+ against the DEPC-induced inhibition of phosphoenzyme formation from Pi. The enzyme was also protected by tight binding of F− and Mg2+against the DEPC-induced inhibition of phosphoenzyme hydrolysis. Peptide mapping of the tryptic digests, detection of peptides containing DEPC-modified histidine by UV absorption at 240 nm, amino acid analysis, sequencing, and mass spectrometry showed that His-5 was a single major residue protected by the above transition state analogs against the modification with DEPC. These results indicate that modification of His-5 with DEPC is responsible for the DEPC-induced inhibition of phosphoenzyme formation from Pi and of phosphoenzyme hydrolysis and suggest that His-5 is located in or very close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step.

intermediate (7,8). A subsequent conformational change of the EP results in Ca 2ϩ release to the lumen (9). Finally, the EP is hydrolyzed to form P i and the dephosphoenzyme. Acetyl phosphate also serves as a substrate through formation and hydrolysis of EP (10,11). The EP can be formed from P i in the presence of Mg 2ϩ and absence of Ca 2ϩ by reversal of EP hydrolysis (12,13). This EP formation occurs through a magnesium-enzyme-phosphate complex that is formed by random binding of Mg 2ϩ and P i to the enzyme (14,15).
It was previously shown (16 -18) that F Ϫ and Mg 2ϩ 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 Mg 2ϩ to form a transition state analog for this EP formation (19,20). By utilizing the protection of the catalytic site by these transition state analogs against chemical modification, we have recently identified Arg-198 involved in 1,2-cyclohexanedione-induced inhibition of EP formation from P i and suggested that this residue is located in or close to the catalytic site in the transition state (21).
It has been well established that modification of histidyl residues in the SR Ca 2ϩ -ATPase by DEPC (22)(23)(24)(25) or photooxidation (22,26,27) causes inhibition of the enzyme. Coan and DiCarlo (24) showed previously that EP hydrolysis in this enzyme is inhibited by modification of histidine with DEPC. However, the histidyl residue(s) involved in this inhibition has not yet been identified.
In the present study, to identify the histidyl residue(s) involved in the DEPC-induced inhibition of the SR Ca 2ϩ -ATPase, we have modified the enzyme with DEPC and examined effects of the above transition state analogs on this modification. DEPC reacts with histidyl residues to yield N-carbethoxyhistidine derivatives (28), which can be detected spectrophotometrically in HPLC of proteolytic digests since this modification causes an increase in absorbance at 240 nm (29,30). We have found that EP formation from P i has been inhibited by modification with DEPC and that the enzyme has been protected by these transition state analogs against this inhibition. Peptide mapping of the tryptic digests, detection of peptides containing DEPC-modified histidine by absorption at 240 nm, amino acid analysis, sequencing, and mass spectrometry have shown that His-5 is a single major residue protected by the transition state analogs against the modification with DEPC. The results indicate that modification of His-5 is responsible for the inhibition of EP formation from P i and suggest that His-5 is located in or very close to the catalytic site in the transition state for EP formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step. * This work was supported by a grant-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

EXPERIMENTAL PROCEDURES
Preparation of SR Vesicles-SR vesicles were prepared from rabbit skeletal muscle and stored at Ϫ80°C as described previously (31). The contents of phosphorylation site in the preparations determined with [␥-32 P]ATP according to Barrabin et al. (32) ranged from 3.9 to 4.4 nmol/mg. Pretreatment of SR Vesicles with F Ϫ and Mg 2ϩ -Pretreatment of the SR vesicles with F Ϫ and Mg 2ϩ was performed as described previously (18) with slight modifications. The vesicles (2 mg/ml) were incubated at 25°C for 3 h in 1 mM KF, 10 mM MgCl 2 , 1 mM EGTA, 0.1 M KCl, 20% (v/v) Me 2 SO, 20% (v/v) glycerol, 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 CaCl 2 , 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 with DEPC-Modification was started at 25°C in 10 mM MgCl 2 , 2 mM CaCl 2 , 0.1 M KCl, and 100 mM MES-NaOH (pH 6.0), unless otherwise stated, by adding DEPC (dissolved in acetonitrile) or the same volume of acetonitrile to a suspension of the SR vesicles to give 10 mg of the vesicles/ml, 4.5 or 0 mM DEPC, and 2% (v/v) acetonitrile. The modification was quenched at 25°C by diluting the mixture 10 times with a solution containing 0.1 M KCl and 7 mM histidine HCl (pH 6.0).
Determination of Tightly Bound F Ϫ and Mg 2ϩ -The SR vesicles were treated with F Ϫ and Mg 2ϩ as described above. In the control samples, CaCl 2 was added to the incubation medium to give 0.1 mM free Ca 2ϩ under the otherwise same conditions as above (tight binding of F Ϫ and Mg 2ϩ is prevented by 0.1 mM Ca 2ϩ (see Ref. 18)). The treated vesicles were washed by centrifugation four times with a solution containing 2 mM EDTA, 0.1 M KCl, 1 M A23187, 10% (v/v) Me 2 SO, and 5 mM MOPS-NaOH (pH 7.0) and suspended in deionized water. Magnesium bound to the vesicles was extracted with 0.8 N HNO 3 , and the concentration of magnesium in the extract was determined by atomic absorption spectrophotometry as described previously (17). The content of tightly bound Mg 2ϩ 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 KNO 3 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 Ϫ was measured by use of a fluoride-selective electrode as described previously (17). The content of tightly bound F Ϫ was obtained by subtracting the content of F Ϫ in the extract from the control sample.
Determination of Bound Vanadate-The SR vesicles (0.2 mg/ml) were incubated at 25°C for 30 min in various concentrations of vanadate, 0.1 M KCl, 20% (v/v) Me 2 SO, 30 mM MOPS-NaOH (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. The concentration of vanadate was measured by the method of Goodno (33). When the SR vesicles were pretreated with F Ϫ and Mg 2ϩ , tightly bound F Ϫ and Mg 2ϩ were entirely released by incubation at 25°C for 30 min in 20 mM CaCl 2 , 0.1 M KCl, and 100 mM MOPS-NaOH (pH 7.0) (17) before vanadate binding was determined.
Proteolysis, Peptide Mapping, Detection of Peptides Containing DEPC-modified Histidine, Amino Acid Analysis, Sequencing, and Mass Spectrometry-The DEPC-modified SR vesicles (5 mg/ml) were digested with TPCK-trypsin (1 mg/ml) at 37°C for 3 h in 20 mM CaCl 2 and 20 mM Tris-HCl (pH 7.0). After centrifugation, the supernatant was subjected to reversed phase HPLC that was performed at a flow rate of 1 ml/min as described previously (34). The absorbance of peptides containing DEPC-modified histidine was monitored at 240 nm (see Refs. 29 and 30), and the absorbance of peptides was monitored at 214 nm. It was difficult to determine the content of DEPC-modified histidine in the DEPC-modified vesicles from the absorbance at 240 nm because, in addition to mono-N-carbethoxyhistidine, bis-carbethoxyhistidine was produced at an unknown ratio to mono-N-carbethoxyhistidine by modification of the vesicles with DEPC (see Fig. 7, Table II, and text described under "Results"). The amino acid compositions of the isolated peptides were analyzed with a precolumn derivatization reversed phase HPLC Waters Pico-Tag system after acid hydrolysis of the peptides in 6 N HCl at 110°C for 21 h. Sequencing was performed with an Applied Biosystems 477A/120A sequencer. Mass determination of the isolated peptides was made on a JEOL JMS-SX102 mass spectrometer with the frit-fast atom bombardment probe as described previously (21).
Phosphorylation of Ca 2ϩ -ATPase-Phosphorylation of the SR vesicles (0.4 mg/ml) with 1 mM 32 P i was performed at 25°C for 10 min in 20 mM MgCl 2 , 5 mM EGTA, 40% (v/v) Me 2 SO, and 50 mM MOPS-Tris (pH 7.0). The reaction was quenched with trichloroacetic acid containing P i . Phosphorylation of the vesicles (1 mg/ml) with 3 mM acetyl [ 32 P]phosphate was performed at 25°C in 5 mM MgCl 2 , 2 mM CaCl 2 , 50 mM KCl, and 50 mM Tris-HCl (pH 7.5), unless otherwise stated. The reaction was quenched with trichloroacetic acid containing nonradioactive acetyl phosphate. The amount of EP formed was determined as described previously (17). When the SR vesicles were pretreated with F Ϫ and Mg 2ϩ , tightly bound F Ϫ and Mg 2ϩ were released as described above before phosphorylation was performed. When the SR vesicles were preincubated with vanadate and Mg 2ϩ , bound vanadate and Mg 2ϩ were entirely released by incubation at 25°C for 30 min in 3 mM CaCl 2 , 2 mM ATP, 0.1 M KCl, and 100 mM MOPS-NaOH (pH 7.0) (35, 36) before phosphorylation was performed.
Miscellaneous Methods-DEPC, TPCK-trypsin, and Na 3 VO 4 were purchased from Sigma. KF was from Nacalai Tesque (Kyoto, Japan). ATP was from Yamasa Biochemicals (Choshi, Japan). Acetyl phosphate was from Kohjin (Tokyo, Japan). [␥-32 P]ATP and 32 P i were obtained from NEN Life Science Products. 32 P i was purified according to Kanazawa and Boyer (12). Acetyl [ 32 P]phosphate was prepared by Procedure B in the method of Stadtman (37). Vanadate solutions were prepared from Na 3 VO 4 according to Goodno (33) just before use. Protein concentrations were determined by the method of Lowry et al. (38) with bovine serum albumin as a standard. Data were analyzed by the nonlinear least squares method as described previously (17). Fig. 1A, when the SR vesicles were pretreated without F Ϫ in the presence of Mg 2ϩ and then treated with DEPC in the presence of 2 mM CaCl 2 (Ⅺ), EP formation from P i was strongly inhibited with apparent first order kinetics at a rate of 0.11 min Ϫ1 and fell to 10% of the original level in 40 min. When the vesicles were pretreated with F Ϫ and Mg 2ϩ and then treated with DEPC (E), the enzyme was partially protected against the DEPC-induced inhibition of EP formation (33.2% maximum inhibition with a rate constant of 0.15 min Ϫ1 ). When the vesicles were treated with DEPC in the presence of 0.2 mM CaCl 2 (छ and Ä) or in the absence of Ca 2ϩ (É and Ç), the DEPC-induced inhibition (छ and É) and the protection by the pretreatment with F Ϫ and Mg 2ϩ (Ä and Ç) were somewhat less than those in the presence of 2 mM CaCl 2 . For this reason, in subsequent experiments, modification with DEPC was performed in the presence of 2 mM CaCl 2 unless otherwise stated. Pretreatment with F Ϫ in the absence of Mg 2ϩ provided no protection (data not shown). Fig.  1B, when the SR vesicles were treated with DEPC in the presence of 0.5 mM vanadate and 10 mM MgCl 2 and absence of Ca 2ϩ (E), the enzyme was protected against the DEPC-induced inhibition of EP formation from P i (49.1% maximum inhibition with a rate constant of 0.12 min Ϫ1 ). The extent of this protection was almost the same as that of the protection afforded by the pretreatment with F Ϫ and Mg 2ϩ . In the absence of vanadate and presence of 9.7 mM Mg 2ϩ (Ⅺ) or in the presence of 0.5 mM vanadate and absence of Mg 2ϩ (Ç), no protection was observed (84.6% maximum inhibition with a rate constant of 0.13 min Ϫ1 ). Fig.  1C, when the SR vesicles were preincubated with P i in the presence of Mg 2ϩ and absence of Ca 2ϩ in 30% (v/v) Me 2 SO at 25°C for 20 min (4.9 nmol/mg EP was formed by this preincubation) and then treated with DEPC, the enzyme was hardly protected against the DEPC-induced inhibition of EP formation from P i (compare E with Ⅺ). Binding of P i to the catalytic site in the presence of 10 mM P i and absence of Mg 2ϩ afforded no protection (compare Ç with É). Binding of Mg 2ϩ in the absence of P i and presence of 9.7 mM Mg 2ϩ provided some protection (compare Ⅺ with É), but higher concentrations of Mg 2ϩ up to 29 mM provided no further protection (data not shown). This is in contrast to no protection afforded by 9.7 mM Mg 2ϩ in the absence of Me 2 SO (see Fig. 1B, Ⅺ).

DEPC-induced Inhibition of Tight Binding of F Ϫ and Mg 2ϩ -
The SR vesicles were treated with or without DEPC for various times, and then tight binding of F Ϫ and Mg 2ϩ was determined (Fig. 2). The amounts of tightly bound F Ϫ and Mg 2ϩ at zero time of the treatment were 17.0 and 8.1 nmol/mg, respectively, being 4.3 and 2.0 times the content of phosphorylation site (4.0 nmol/mg) in the vesicles used. This is in agreement with our previous findings (18,21) that the stoichiometry of tight binding of F Ϫ and Mg 2ϩ to the maximum level of phosphorylation is 4:2:1. The tight binding was inhibited progressively during the FIG. 1. DEPC-induced inhibition of EP formation from P i , protection by pretreatment with F ؊ and Mg 2؉ or by presence of vanadate and Mg 2؉ against the inhibition, and lack of protection by EP formation from P i against the inhibition. A, the SR vesicles were pretreated with (E, q, Ä, ä, Ç, and å) or without (Ⅺ, f, छ, ࡗ, É, and ç) KF in the presence of MgCl 2 , otherwise as described under "Experimental Procedures." The vesicles were then incubated with (E, Ⅺ, Ä, छ, Ç, and É) or without (q, f, ä, ࡗ, å, and ç) DEPC for various times in the presence of 2 mM CaCl 2 (E, q, Ⅺ, and f), 0.2 mM CaCl 2 (Ä, ä, छ, and ࡗ), or 5 mM EGTA without added CaCl 2 (Ç, å, É, and ç), otherwise as described under "Experimental Procedures." EP formation from 32  . The amount of EP formed from P i by the preincubation in the presence of P i , MgCl 2 , and EGTA (E and q) was 4.8 nmol/mg. After addition of acetonitrile with (E, Ⅺ, Ç, and É) or without (q, f, å, and ç) DEPC, the vesicles were incubated for various times. The final composition was 10 mg of vesicles/ml, 0 or 4.5 mM DEPC, 0, 2, or 10 mM P i , 0 or 10 mM MgCl 2 (9.7 mM Mg 2ϩ ), 0 mM CaCl 2 , 0 or 10 mM EDTA, 0 or 10 mM EGTA, 30% (v/v) Me 2 SO, 2% (v/v) acetonitrile, and 100 mM MES-NaOH (pH 6.0). EP formation from 32 P i was determined. Solid and dashed lines show least squares fit to single exponentials. treatment with DEPC (E and Ç). The rate of this inhibition (0.10 min Ϫ1 ) was in agreement with that of the DEPC-induced inhibition of EP formation from P i , although the maximum inhibition (72-73%) appeared to be slightly less than that of the EP formation (see Fig. 1A, Ⅺ).
DEPC-induced Inhibition of Vanadate Binding and Protection by Pretreatment with F Ϫ and Mg 2ϩ against the Inhibition-The SR vesicles were pretreated with or without F Ϫ in the presence of Mg 2ϩ and then treated with or without DEPC for 40 min (Fig. 3A). After tightly bound F Ϫ and Mg 2ϩ were removed, vanadate binding was determined at various concentrations of vanadate in the presence (Ç, Ⅺ, and E) and absence (å, f, and q) of Mg 2ϩ . In the control (Ç), in which the vesicles were pretreated without F Ϫ and then treated without DEPC, vanadate binding in the presence of Mg 2ϩ increased with increasing concentration of vanadate and was saturated with 4 M vanadate. The maximum level of this binding was in approximate agreement with the content of phosphorylation site (4.0 nmol/mg). This binding was nearly completely inhibited by the treatment with DEPC (Ⅺ). When the vesicles were pretreated with F Ϫ and Mg 2ϩ and then treated with DEPC (E), the enzyme was strongly protected against the DEPC-induced inhibition of vanadate binding.
When the vesicles were pretreated without F Ϫ and then treated with DEPC for various times (Fig. 3B, Ⅺ), vanadate binding in the presence of Mg 2ϩ was inhibited with apparent first order kinetics at a rate of 0.11 min Ϫ1 , and its maximum inhibition was 80.4%. These kinetic parameters were in essential agreement with the rate (0.11 min Ϫ1 ) and maximum (89.7%) of the DEPC-induced inhibition of EP formation from P i under the same conditions (see Fig. 1A, Ⅺ). The enzyme was protected by the pretreatment with F Ϫ and Mg 2ϩ against the inhibition of vanadate binding (Fig. 3B, E) to a similar extent (44.2% maximum inhibition with a rate constant of 0.09 min Ϫ1 ) as protected against the inhibition of EP formation from P i (see Fig. 1A, E).
DEPC-induced Weak Inhibition of EP Formation from Acetyl Phosphate-The SR vesicles were pretreated with (E and q) or without (Ⅺ and f) F Ϫ in the presence of Mg 2ϩ (Fig. 4, A and B).
In the experiments shown in Fig. 4A, the pretreated vesicles were treated with (E and Ⅺ) or without (q and f) DEPC for various times. After tightly bound F Ϫ and Mg 2ϩ were removed, EP formation from acetyl phosphate was determined. DEPC caused only weak inhibition of the EP formation when the vesicles were pretreated without F Ϫ (compare Ⅺ with f). This inhibition was unaffected by the pretreatment with F Ϫ and Mg 2ϩ (compare E with Ⅺ). In contrast to the results reported by Coan and DiCarlo (24), EP formation from ATP was almost completely inhibited by the treatment with DEPC (data not shown). This discrepancy may be possibly due to the difference in the experimental conditions used.
In the experiments shown in Fig. 4B, the pretreated vesicles were treated with (E and Ⅺ) or without (q and f) DEPC for 40 min. After tightly bound F Ϫ and Mg 2ϩ were removed, the vesicles were phosphorylated with acetyl phosphate for various times. When the vesicles were pretreated without F Ϫ and then treated with DEPC (Ⅺ), EP formation was only slightly slower than that (f) in the vesicles that were pretreated without F Ϫ and then treated without DEPC. The pretreatment with F Ϫ and Mg 2ϩ had virtually no effect on the kinetics of EP formation in the DEPC-treated vesicles (compare E with Ⅺ). Fig. 4C, the SR vesicles were pretreated with F Ϫ and Mg 2ϩ (E and q), without F Ϫ and with Mg 2ϩ (Ⅺ and f), or with F Ϫ and without Mg 2ϩ (Ç and å) and then treated with (E, Ⅺ, and Ç) or without (q, f, and å) DEPC for 40 min. After tightly bound F Ϫ and Mg 2ϩ were removed, the vesicles were phosphorylated with acetyl [ 32 P]phosphate at 0°C for 4 min. EP formation was quenched by addition of nonradioactive acetyl phosphate and EGTA, and the decay of EP was followed. When the vesicles were treated without DEPC (q, f, and å), the EP decay was rapid and could be described by a single exponential with a decay constant of 2.37 min Ϫ1 . The kinetics of this decay was unaffected by any above pretreatment. When the vesicles were pretreated without F Ϫ and with Mg 2ϩ (Ⅺ) or with F Ϫ and without Mg 2ϩ (Ç) and then treated with DEPC, the EP decay was very slow and could be described by a single exponential with a decay constant of 0.17-0.21 min Ϫ1 . When the vesicles were pretreated with F Ϫ and Mg 2ϩ and then treated with DEPC (E), the EP Partial Restoration of EP Formation from P i by the Treatment of DEPC-modified SR Vesicles with Hydroxylamine-The SR vesicles were pretreated with or without F Ϫ in the presence of Mg 2ϩ and then treated with or without DEPC for 12 min. After tightly bound F Ϫ and Mg 2ϩ were removed, the vesicles were treated with 0.5 M hydroxylamine (pH 7.0) or with 0.5 M NaCl, and then EP formation from P i was determined (Table I). When the vesicles were pretreated without F Ϫ , EP formation from P i was markedly inhibited by the treatment with DEPC. The EP formation was partially restored by the subsequent treatment with hydroxylamine. The treatment with NaCl substituted for hydroxylamine caused no restoration of EP formation. When the vesicles were pretreated with F Ϫ and Mg 2ϩ , the DEPC-induced inhibition of EP formation was suppressed considerably. The subsequent treatment with hydroxylamine again caused a partial restoration of EP formation.

DEPC-induced Inhibition of Hydrolysis of EP Formed from Acetyl Phosphate-In the experiments shown in
Peptide Mapping of Tryptic Digests of DEPC-modified SR Vesicles-In the first series of experiments (Fig. 5, A-C), the SR vesicles were pretreated without F Ϫ and with Mg 2ϩ (A), with F Ϫ and without Mg 2ϩ (B), or with F Ϫ and Mg 2ϩ (C), and then treated with DEPC. The vesicles were digested with TPCKtrypsin and subjected to reversed phase HPLC. The peptide maps at 214 nm (lower traces of A-C) agreed closely with each   In the second series of experiments (Fig. 5, D-G), the SR vesicles were treated with DEPC in the absence of vanadate and Mg 2ϩ (D), in the absence of vanadate and presence of Mg 2ϩ (E), in the presence of vanadate and absence of Mg 2ϩ (F), or in the presence of vanadate and Mg 2ϩ (G). In reversed phase HPLC of the tryptic digests, the peaks indicated by arrows I-III were appreciably reduced only when both vanadate and Mg 2ϩ were present during the treatment with DEPC (compare upper trace of G with upper traces of D-F).
In the third series of experiments (Fig. 5, H-K It is clear from the chromatographic profiles that the peaks reduced by the presence of vanadate and Mg 2ϩ (Fig. 5, D-G,  arrows) or by the presence of Mg 2ϩ and Me 2 SO (Fig. 5, H-K, arrows) corresponded to those reduced by the pretreatment with F Ϫ and Mg 2ϩ (Fig. 5, A-C, arrows).
Although the changes of peak III in different conditions were very small (but reproducible) in these experiments, the extent of the reduction in this peak varied with different preparations of SR vesicles used. In fact, in the experiments shown in Fig. 6  (A and B), the reduction in peak III was substantial.
Purification of DEPC-modified Peptides-The SR vesicles were pretreated without (Fig. 6A) or with (Fig. 6B) F Ϫ in the presence of Mg 2ϩ and then treated with DEPC. The vesicles were digested with TPCK-trypsin and subjected to the first reversed phase HPLC.
Fractions I-III in A and B were pooled separately and subjected to the second reversed phase HPLC (data not shown). Fraction I from A gave a peak at 240 nm that was strongly reduced in the second HPLC of fraction I from B. Fraction II from A gave a major peak at 240 nm that was also greatly reduced in the second HPLC of fraction II from B. Fraction III from A gave a peak at 240 nm that was again greatly reduced in the second HPLC of fraction III from B. Fractions IV from A and B showed no difference in the second HPLC. The peaks sensitive to the pretreatment with F Ϫ and Mg 2ϩ were further purified by the third reversed phase HPLC (data not shown). Purified peptides, Peptide 1 (from fraction I in A), Peptide 2 (from fraction II in A), and Peptide 3 (from fraction III in A), were obtained by final reversed phase HPLC (Fig. 6, C-E).
The peak with retention time of about 47 min in Fig. 6A was also reduced by the pretreatment with F Ϫ and Mg 2ϩ (Fig. 6B). However, this peak was not analyzed since the reduction in this peak was not reproducible.
Hydroxylamine Sensitivity of Isolated DEPC-modified Peptides-The peptides isolated as above were analyzed by reversed phase HPLC before and after treatment with 0.5 M hydroxylamine (pH 7.0). Hydroxylamine-treated Peptides 1 and 2 had the same shorter retention time and a much smaller A 240 nm /A 214 nm ratio than untreated Peptides 1 and 2 (data not shown). The absorption spectra of untreated Peptides 1 and 2 showed shoulders at 240 nm (Fig. 7, A and B, solid lines), which are characteristic of mono-N-carbethoxyhistidine (28). These shoulders disappeared when the peptides were treated with hydroxylamine (Fig. 7, A and B, dashed lines). These results suggest the presence of mono-N-carbethoxyhistidine in Peptides 1 and 2. In contrast, treatment of Peptide 3 with hydroxylamine caused no change in the reversed phase HPLC profile and in the absorption spectrum (Fig. 7C, solid and dashed  lines). Peptide 3 had a 1.8 times higher A 240 nm /A 214 nm ratio than Peptides 1 and 2, but its absorption spectrum showed no definite shoulder at 240 nm. These results suggest the presence of bis-carbethoxyhistidine in Peptide 3 (see Ref. 28).
Amino Acid Analysis, Sequencing, and Mass Analysis of Isolated DEPC-modified Peptides-Peptides 1 and 2 had the same amino acid composition (Table II) that exactly matched the amino-terminal 7-amino acid sequence (Met-Glu-Ala-Ala-His-Ser-Lys) in the SR Ca 2ϩ -ATPase. Peptide 3 also had the same amino acid composition except that histidine was missing. This missing of histidine is consistent with the above idea that histidine of Peptide 3 is bis-carbethoxylated, because it is known (28) that acid hydrolysis in amino acid analysis does not regenerate histidine from bis-carbethoxyhistidine although it regenerates histidine from mono-N-carbethoxyhistidine. Attempts to sequence these three peptides were unsuccessful, indicating that they were blocked on their amino termini. This again suggests that these peptides are the amino-terminal peptides of the SR Ca 2ϩ -ATPase, because the amino terminus of this enzyme is blocked by an acetyl group (39). Masses of Peptides 1 and 2 were both 887.5, being in good agreement with the monoisotopic mass (887.4) of the above acetylated aminoterminal peptide calculated on the assumption that His-5 is mono-N-carbethoxylated. Mass of Peptide 3 was 949.4, being in exact agreement with the monoisotopic mass (949.4) of the above acetylated peptide calculated on the assumption that His-5 is bis-carbethoxylated. These results lead to the conclusion that His-5 in the SR Ca 2ϩ -ATPase was modified with DEPC and that this modification was specifically inhibited by the pretreatment with F Ϫ and Mg 2ϩ (Fig. 5, A-C, arrows), by the presence of vanadate and Mg 2ϩ (Fig. 5, D--G, arrows), or by the presence of Mg 2ϩ and Me 2 SO (Fig. 5, H--K, arrows). The reason why peptides with the same sequence and same modification gave different retention times in HPLC remains obscure.  Fig. 1C. The vesicles were then incubated with DEPC for 40 min, otherwise as in Fig. 1C. In A-K, the vesicles were washed by centrifugation with a solution containing 20 mM CaCl 2 and 20 mM Tris-HCl (pH 7.0), digested with TPCK-trypsin, and subjected to reversed phase HPLC. Elution was performed with the following linear gradient of acetonitrile in 3.7 mM potassium phosphate buffer (pH 6.4): 0% from 0 to 30 min, 35% at 90 min, and 70% at 100 min. The absorbance at 214 nm (lower traces) and the absorbance at 240 nm (upper traces) were monitored.

DISCUSSION
The observed protection by tight binding of F Ϫ and Mg 2ϩ (Fig. 1A) or by high affinity binding of vanadate in the presence of Mg 2ϩ (Fig. 1B) against the DEPC-induced inhibition of EP formation from P i suggests that a histidyl residue(s) protected by these transition state analogs contributes toward formation of the transition state for EP formation from the magnesiumenzyme-phosphate complex or, alternatively, that the protected histidyl residue(s) is located very close to the essential compo-nents (bound phosphate, bound Mg 2ϩ , or other functional groups) of this transition state. This view is consistent with the findings that binding of these transition state analogs is inhibited by the modification with DEPC (Figs. 2 and 3) and that the enzyme is protected by tight binding of F Ϫ and Mg 2ϩ against the DEPC-induced inhibition of high affinity vanadate binding (Fig. 3). It is also in harmony with the observed lack of protection by formation of EP from P i or by formation of the enzymephosphate complex (Fig. 1C), because it is likely that the struc- Fractions I, II, and III from A and B were subjected to the second reversed phase HPLC. Elution was performed with linear gradients of acetonitrile in 20 mM Na 2 SO 4 and 5 mM sodium phosphate (pH 6.4). Fractions sensitive to the pretreatment with F Ϫ and Mg 2ϩ were pooled separately and subjected to the third reversed phase HPLC. Elution was performed with a linear gradient of acetonitrile in 0.1% ammonium trifluoroacetate (pH 6.4). Fractions sensitive to the pretreatment with F Ϫ and Mg 2ϩ were further subjected to final reversed phase HPLC (C-E). Elution was performed with the following linear gradients of acetonitrile in 0.1% ammonium trifluoroacetate (pH 6.4): C, 0% from 0 to 30 min, 9% at 35 min, 13.5% at 75 min, and 90% at 80 min; D, 0% from 0 to 30 min, 9% at 35 min, 18% at 75 min, and 90% at 80 min; E, 0% from 0 to 30 min, 11.3% at 35 min, 20.3% at 75 min, and 90% at 80 min. Peptides 1, 2, and 3 thus isolated were subjected to amino acid analysis, sequencing, and mass spectrometry. A-E, the absorbance at 214 nm (lower traces) and the absorbance at 240 nm (upper traces) were monitored.
tures of the stable intermediates (the enzyme-phosphate complex and EP) are different from the structure of the transition state for EP formation from the magnesium-enzymephosphate complex.
The reduction in three specific peaks by tight binding of F Ϫ and Mg 2ϩ (Fig. 5C, arrows) or by high affinity binding of vanadate in the presence of Mg 2ϩ (Fig. 5G, arrows) indicates that a few specific histidyl residues are selectively protected by these transition state analogs against the modification with DEPC. The data from peptide purification (Fig. 6), amino acid analysis (Table II), sequencing, and mass spectrometry show that His-5 is a single major histidyl residue protected by these analogs against the modification. These findings indicate that modification of His-5 with DEPC is responsible for the DEPCinduced inhibition of EP formation from P i (Fig. 1, A and B) and further suggest that His-5 is located in or very close to the catalytic site in the transition state for EP formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step.
The limited protection by Mg 2ϩ in the presence of Me 2 SO against the DEPC-induced inhibition of EP formation from P i (Fig. 1C) and against the modification of His-5 with DEPC (Fig.  5I, arrows) suggests that His-5 is located close to the Mg 2ϩbinding site in the magnesium-enzyme complex. The lack of protection by Mg 2ϩ in the absence of Me 2 SO (Fig. 1B and Fig.  5E, arrows) may be possibly due to lower affinity for Mg 2ϩ in the absence of Me 2 SO (40). The lack of protection by the presence of 10 mM P i in the absence of Mg 2ϩ against the DEPCinduced inhibition of EP formation from P i (Fig. 1C) and against the modification of His-5 with DEPC (Fig. 5J, arrows) indicates that His-5 is not located at the P i -binding site in the enzyme-phosphate complex.
The weak inhibition of EP formation from acetyl phosphate by the modification with DEPC (Fig. 4A), the lack of protection by tight binding of F Ϫ and Mg 2ϩ against this inhibition (Fig.  4A), and the lack of an appreciable effect of the modification with DEPC on the kinetics of EP formation from acetyl phosphate (Fig. 4B) indicate that phosphoryl transfer from acetyl phosphate to the phosphorylation site in the forward reaction is not substantially affected by the modification of His-5.
The DEPC-induced inhibition of hydrolysis of EP formed from acetyl phosphate (Fig. 4C) is consistent with the inhibition of EP formation from P i (Fig. 1, A and B), because EP hydrolysis is reversal of EP formation from P i . However, we cannot exclude the possibility that the conformational change of EP immediately preceding its hydrolytic cleavage is also inhibited by this modification.
Biphasic kinetics of the EP decay shown in Fig. 4C (dashed line drawn under open circles) is interpreted in terms of the presence of the fast and slow decaying populations that comprise EP having unmodified His-5 and DEPC-modified His-5, respectively. This interpretation is supported by the findings that the decay constants for the fast and slow decaying components are in good agreement with the decay constants for EP formed with the unmodified vesicles (Fig. 4C, dotted line drawn under solid symbols) and with the unprotected DEPC-modified vesicles (Fig. 4C, solid and dash-dotted lines drawn under open  squares and open triangles), respectively. The results indicate that the rate of EP hydrolysis is reduced to less than 10% by the modification of His-5 with DEPC and further suggests that the observed inhibition of EP hydrolysis and of its reversal is almost exclusively due to the modification of His-5 with DEPC.
His-5 is conserved in sarco(endo)plasmic reticulum Ca 2ϩ -ATPases (1, 41-43) but not in plasma membrane Ca 2ϩ -ATPase (44). Therefore, we cannot entirely exclude the possibility that the observed inhibition is due to steric hindrance induced by DEPC-modified His-5.
The secondary structural model for the Ca 2ϩ -ATPase suggests that the enzyme is composed of 10 transmembrane ␣helices (M 1 to M 10 ) and a cytoplasmic globular fraction, which is divided into two main domains, a small cytoplasmic loop between M 2 and M 3 and a large cytoplasmic loop between M 4 and M 5 (1). The large cytoplasmic loop contains the phosphorylation site (1, 4 -6) and the ATP-binding site (34,(45)(46)(47)(48)(49). The functional role of the small cytoplasmic loop is less clear, but we FIG. 7. Hydroxylamine sensitivity of isolated DEPC-modified peptides. A part of each of isolated Peptides 1, 2, and 3 was treated with 0.5 M hydroxylamine (pH 7.0) at 25°C for 30 min. The treated and untreated peptides were analyzed by reversed phase HPLC. Elution was performed with the following linear gradients of acetonitrile in 3.7 mM sodium phosphate buffer (pH 6.4): A and B, 0% from 0 to 20 min, 14% at 60 min, and 70% at 70 min; C, 0% from 0 to 20 min, 21% at 60 min, and 70% at 70 min. The absorbance at 214 nm and the absorbance at 240 nm were monitored. Solid lines show absorption spectra of untreated Peptides 1 (A), 2 (B), and 3 (C), which were obtained by spectrum scanning after the flows were stopped at the peaks of absorption at 240 nm and then by subtracting background spectra of the eluting solutions at the peaks. Dashed lines show absorption spectra of treated Peptides 1 (A), 2 (B), and 3 (C), which were obtained at the peaks of absorption at 240 nm in the same way as above. have recently suggested that Arg-198 in this loop is located in or close to the catalytic site in the transition state for EP formation from the magnesium-enzyme-phosphate complex (21). The amino-terminal region preceding the first transmembrane helix (M 1 ) is known to be exposed to the cytoplasm (50). Its functional role is unknown, although it was previously shown (51) that deletion of the amino-terminal 30 amino acids inhibits the stable insertion of the enzyme into the membrane and inactivates the enzyme. Our present results suggest that the amino-terminal domain including His-5 contributes to the catalytic site.