INTRODUCTION

The sarcoplasmic reticulum (SR)¹ Ca²⁺-ATPase is a 115-kDa transmembrane protein that couples ATP hydrolysis to removal of Ca²⁺ from the cytosol in skeletal muscle fibers, triggering the end of muscular contraction (Hasselbach and Makinose, 1961; Ebashi and Lipmann, 1962). The primary structure of this enzyme has been determined and has led to predictions of secondary structure and enzyme topology (Brandl et al., 1986; Clarke et al., 1989). Molecular imaging of ATPase crystals seems to agree with general structural predictions (Stokes et al., 1994), but questions concerning the role of specific amino acids in binding and transporting Ca²⁺ as well as the structure of the nucleotide binding site(s) remain unanswered. Efforts to map the enzyme functionally have used chemical modification by specific reagents and site-specific mutagenesis. This latter technique has been used recently to identify amino acids that affect kinetic parameters related to the catalytic site and to the Ca²⁺ binding sites (Maruyama and MacLennan, 1988; Maruyama et al., 1989; Clarke et al., 1990; Vilsen et al., 1991). In addition, many amino acids have been labeled, identified, and located within the primary structure using a variety of probes for the nucleotide binding site. These experiments have almost always involved probes that bind covalently to the enzyme, such as FITC (Mitchinson et al., 1982), 8-azido-ATP (Lacapère et al., 1993), pyridoxal 5-phosphate (Yamagata et al., 1993), oxidized ATP (Mignaco et al., 1990), or chemical reagents that cause radical structural changes, such as glutaraldehyde (McIntosh, 1992) or dithiothreitol (Daiho and Kanazawa, 1994). Thus, kinetic parameters such as those involved in enzyme phosphorylation and dephosphorylation could not always be assessed with the labeled enzyme, and the precise functional role of the labeled amino acids remains uncertain.

It has been reported that Ca²⁺-ATPase modified with FITC loses all the activities related to ATP binding (Pick, 1981; Pick and Bassilian, 1981). Because Lys-515, which is the point of attachment of FITC to the enzyme, is neither unusually reactive toward FITC (Murphy, 1988) nor essential for ATP hydrolysis (Maruyama et al., 1989), it has been suggested that it is the fluorescein moiety of FITC that directs the reaction toward the ATP site. Thus, affinity binding of fluorescein would bring the isothiocyanate group close to Lys-515, promoting a specific attachment, and fluorescein would then remain anchored to the putative ATP binding site, thus leading to inhibition of the Ca²⁺-ATPase activity.

In this study we use erythrosin B, a halogenated derivative of fluorescein that is considered to bind to enzyme nucleotide binding sites with high affinity and specificity (Lundblad and Noyes, 1984; Neslund et al., 1984), to photo-oxidize residues on SR Ca²⁺-ATPase, and we examine its influence on the partial reactions of the hydrolytic cycle. Erythrosin is capable of promoting selective photo-oxidation of amino acids, which can be useful in the identification of groups involved in catalysis (Lundblad and Noyes, 1984; Neslund et al., 1984; Halliwell and Gutteridge, 1989), and has two advantages: even after photo-oxidation the sites would still be able to bind ATP, and no bulky group would remain covalently bound to the enzyme.

MATERIALS AND METHODS

Sarcoplasmic reticulum vesicles were obtained from rabbit hind leg skeletal muscle as described by Eletr and Inesi (1972). Ca²⁺-ATPase was purified by method number 2 of Meissner et al. (1973) and stored in liquid nitrogen. Protein determinations were performed according to Lowry et al. (1951), using bovine serum albumin as standard.

Unless otherwise stated, Ca²⁺-ATPase was incubated with the dyes for 10 min at 25 °C under room light in medium containing 20 mM Tris-HCl (pH 7.4), 0.05 mM CaCl₂, 80 mM KCl, 5 mM MgCl₂, and the dye concentrations specified in the legends before starting the reactions. The protein concentration on the assays could vary from 10 to 50 µg/ml. In this range there was no significant variation in the sensitivity of the ATPase toward photo-inhibition by erythrosin. However, when the protein concentration was raised to 0.2 mg/ml for the experiments of phosphorylation by P_i, approximately three times as much erythrosin was needed to reach the same extent of inhibition.

ATPase activities were assayed at 37 °C in reaction medium containing 20 mM Tris-HCl (pH 7.4), 0.05 mM CaCl₂, 80 mM KCl, 5 mM MgCl₂, 10-20 µg/ml purified Ca²⁺-ATPase, and 2 mM ATP. In most cases, activity was determined by counting the amount of radioactive P_i released from [-³²P]ATP. The reaction was quenched and free nucleotide was trapped by the addition of an equal volume of activated charcoal suspended in 0.1 N HCl (Grubmeyer and Penefsky, 1981). After centrifugation the P_i remaining in the supernatant was counted in a liquid scintillation counter. In some experiments, release of nonradioactive P_i was measured using molybdovanadate reagent (Lin and Morales, 1977).

The differential absorbance spectrum for control versus erythrosin-treated samples was recorded using a Milton-Roy Spectronic 3000 Array Spectrophotometer. The samples contained 0.2 mg/ml Ca²⁺-ATPase, 1 µM erythrosin, 20 mM Tris-HCl (pH 7.4), 80 mM KCl, 5 mM MgCl₂, and 50 µM CaCl₂. The absorbance spectra were recorded before photo-oxidation and then after a 60-min incubation period, either in the presence or the absence of light, and the differential absorbance spectrum was calculated.

Steady-state Phosphorylation of Ca²⁺-ATPase with [-³²P]ATP

Ca²⁺-ATPase (20-50 µg/ml) was incubated at 25 °C in medium with the same composition used to measure ATPase activity but using 15 µM [-³²P]ATP. After photo-oxidation, phosphorylation of the enzyme was started by the addition of [-³²P]ATP, and the reaction was quenched after 3 s by adding 10 volumes of an ice-cold solution of 250 mM perchloric acid and 5 mM P_i. Samples were then filtered through Millipore filters (0.45-µm pore size) and washed five times with the same volume of quenching solution. The amount of radioactivity on the filters was counted in a liquid scintillation counter, and phosphoenzyme was determined according to Knowles and Racker (1975).

Ca²⁺-ATPase (0.1 mg) was phosphorylated in 0.5 ml of a medium containing 20 mM MES-NaOH (pH 6.0), 1 mM EGTA, 10 mM MgCl₂, and 4 mM [³²P]P_i. After 5 min, the reaction was quenched by adding 10 volumes of ice-cold 250 mM perchloric acid and 5 mM P_i. The resulting samples were treated as described above to measure incorporated [³²P]P_i.

The formation or decay of phosphoenzyme was followed using a rapid mixing device coupled to either two or three syringes. For phosphorylation experiments, one syringe contained Ca²⁺-ATPase (40 µg/ml), 20 mM Tris-HCl (pH 7.4), 80 mM KCl, 5 mM MgCl₂, 0.2 mM EGTA, and varying erythrosin, whereas a second syringe contained 20 mM Tris-HCl (pH 7.4), 80 mM KCl, 30 µM [-³²P]ATP, 5 mM MgCl₂, 0.3 mM CaCl₂. In some experiments, both syringes contained 0.05 mM CaCl₂, and the same concentration of erythrosin. The protein solution of syringe 1 was preincubated for 10 min at 25 °C under room light before initiating the phosphorylation reactions. The solutions were forced through a mixing chamber connected to a capillary tube, whose length was varied to obtain different reaction times. This capillary emptied into a quenching tube containing 5 volumes of a mixture of 250 mM perchloric acid and 5 mM P_i. For dephosphorylation experiments, the capillary was connected to a second mixing chamber, to which a third syringe delivered the dephosphorylation solution (a volume equivalent to 88% of the sum of volumes of syringes 1 and 2). Syringe 3 contained 22.8 mM Tris-HCl (pH 7.4), 91.2 mM KCl, 5.7 mM MgCl₂, 6.84 mM EGTA, 0 or 2.28 mM ADP, and 1.14 times the dye concentration used in syringes 1 and 2. Reactions were quenched in acid, and phosphorylated protein samples were treated as above.

Free Ca²⁺ concentrations were calculated with the Ca²⁺-EGTA dissociation constants of Schwarzenbach et al. (1957), using an algorithm developed by Fabiato and Fabiato (1979). Reaction media were the same as that used for ATPase activity measurements, except that 2 mM CaCl₂ and various EGTA concentrations were added.

Ca²⁺ binding to control and erythrosin-photo-oxidized Ca²⁺-ATPase was measured in medium containing 10 µM [⁴⁵Ca]CaCl₂, 50 µg/ml Ca²⁺-ATPase, 2 mM ATP, 20 mM Tris-HCl (pH 7.4), 80 mM KCl, and 5 mM MgCl₂. Reactions were stopped by Millipore filtration, and the retained radioactivity was counted. Blanks obtained either by processing the reaction at pH 5.0 or by omission of the enzyme were discounted to correct for nonspecific binding of [⁴⁵Ca]Ca²⁺ to the enzyme and for the dead volume of the filters, respectively. Both blanks were identical, and the radioactivity corresponding to specific binding was always equivalent to approximately twice the radioactivity of the blanks.

After photo-oxidation with 1 µM erythrosin for 60 min, the Ca²⁺-ATPase was further incubated with glutaraldehyde for 90 min in the presence of 0-100 µM ADP as described by McIntosh (1992). The medium contained 0.2 mg/ml Ca²⁺-ATPase, 20 mM MOPS-Tris (pH 8.1), 80 mM KCl, 5 mM MgCl₂, 0.05 mM CaCl₂, and 0.142 mM glutaraldehyde. The cross-linking reaction was quenched with one volume of a denaturing buffer containing 2% SDS, 8 M urea, 200 mM Tris-HCl (pH 6.8), and 500 mM 2-mercaptoethanol, and submitted to SDS-polyacrylamide gel electrophoresis (Laemmli, 1970). In another set of experiments, photo-oxidation was omitted, and ADP was replaced by equivalent concentrations of erythrosin.

Fluorescence was measured at room temperature in a continuously stirred 1-cm light path quartz cuvette using a Hitachi F-3010 spectrofluorimeter. Excitation was set at 408 nm, and emission was set at 540 nm. The emission values were corrected for erythrosin fluorescence and filter effects.

Kinetic reactions were simulated in an IBM PC computer, using a program described by Hecht et al. (1990), modified with the introduction of input-output subroutines. The program, based in an optimization of the fourth order Runge-Kutta-Gill algorithm, was run using an integration time interval of 1 × 10⁵ s.

ATP and ADP (sodium salts), 2-mercaptoethanol, glutaraldehyde, Tris, MOPS, and EGTA were from Sigma; fluorescein (free acid or sodium salt), erythrosin B, and eosin Y were from Riedel; [⁴⁵Ca]Ca²⁺ was from DuPont NEN; [³²P]P_i was purchased from IPEN (São Paulo-Brasil) and purified on a Dowex 50-W column according to Kessler et al. (1986). [-³²P]ATP was prepared according to Walseth and Johnson (1979). All other reagents were of analytical grade.

RESULTS

After a 10-min incubation with the enzyme, erythrosin B proved to be the best photo-inhibitor when compared with fluorescein and eosin Y (Fig. 1A). Erythrosin presented the lowest K_0.5 (0.5-1.0 µM), followed by eosin (K_0.5 of 4-5 µM) and fluorescein (K_0.5 > 100 µM). The extent of inhibition by erythrosin increased with the time of exposure to light and also increased with dye concentration. The addition of ATP (Fig. 1B) or ADP (not shown) during exposure to light partially protected the enzyme against photo-oxidation with erythrosin. The photo-inhibition elicited by erythrosin was irreversible, because several centrifugation-washing cycles or dilution of the phospholipids with large excess of detergents such as polydocanol or C₁₂E₈ did not restore the ATPase activity (not shown). Erythrosin (and eosin, not shown) also induced the inhibition of ATP hydrolysis in the absence of light. The K_0.5 for this effect was 5-10-fold higher than in the light (Fig. 1C). Unlike the photo-inhibition, the inhibition in the dark was not time-dependent and was almost fully reversible after the washing or detergent treatments indicated above (not shown).To determine whether the protein had indeed been covalently modified by photo-oxidation with erythrosin, we measured the differential absorbance of the protein samples prior to and after reaction in the light. The differential absorption spectrum resulting from the photo-oxidation of Ca²⁺-ATPase with erythrosin is shown in Fig. 2. This spectrum agrees with the previous observations of Stuart et al. (1992) for the photo-oxidation of heavy sarcoplasmic reticulum vesicles with rose bengal and strongly suggests that the inhibition in the light is the result of a modification of some amino acid residue of the enzyme, because in the absence of light no variation in absorbance of the samples was detected. However, the photo-oxidation did not significantly modify the isoelectric point of the protein, as revealed by isoelectric focusing of the normal and oxidized protein and by nonionic slab-gel polyacrylamide gel electrophoresis (data not shown).

The ATP concentration dependence of Ca²⁺-ATPase activity was studied using low erythrosin concentrations (0.2 and 0.5 µM) for photo-inhibition in order to achieve measurable ATP hydrolysis together with observable kinetic effects. The photo-inhibition of the ATPase activity induced by erythrosin was not reversed by increasing the ATP concentration. Both V_max1 and V_max2 were significantly and proportionally reduced, with no change in the affinity for ATP at the catalytic site. However, a significant decrease was observed in the apparent affinity for the secondary activation induced by ATP. These data are summarized in Table I. A similar inhibitory behavior with erythrosin was observed for the erythrocyte Ca²⁺-ATPase by Mugica et al. (1984). Those authors concluded that inhibition with erythrosin was noncompetitive with ATP for the catalytic site but that the dye modified the affinity and competed with ATP for the regulatory site of the enzyme.

Table I. Kinetic parameters of the Ca2+-ATPase photo-inhibited with erythrosin The values are calculated from the best fit to the experimental points, considering a biphasic response to the ATP concentration using the nonlinear regression program Enzfitter. The curve was generated assuming a model of two interconverting ATP binding sites, one with high affinity that behaves as the catalytic site and one with low affinity that acts as the regulatory site. The rates and affinities for ATP hydrolysis are calculated according to the equation v = (Vm1/[1 + Km1/S + S/Km2] + (Vm2/[1 + {1 + Km1/S} × Km2/S]), where Vm1 is the maximal velocity at the catalytic site and Vm2 is the velocity calculated for the sum of the effects of both sites. Erythrosin Km1 Vm1 Km2 Vm2 µM µM µmol/mg/min µM µmol/mg/min 0 1.17 1.33 500 10.5 0.2 1.00 1.11 833 9.0 0.5 1.11 0.91 1250 7.7

The affinity of the enzyme for Ca²⁺ was not modified by photo-inhibition, considering both the high and low affinity Ca²⁺ binding sites, although the apparent cooperativity may have been slightly decreased (Fig. 3). Equilibrium binding of Ca²⁺ to the photo-oxidized enzyme reached the same values as in the control, attaining approximately 8 nmol of [⁴⁵Ca]Ca²⁺ bound per milligram of Ca²⁺-ATPase with 10 µM Ca²⁺ in the medium. This result rules out the possibility that the observed decrease in ATPase activity could be due to destruction of the Ca²⁺ binding sites transforming the inhibited enzyme into an incompetent silent form. It also establishes that inhibition is not a result of nonsaturation of these sites due to a decrease in affinity.

The same steady-state values for phosphorylation with ATP were obtained with control enzyme and photo-inhibited Ca²⁺-ATPase after illumination with 5 µM erythrosin during 10 min. Because ATPase activity was inhibited up to 80% in these conditions, this observation indicates that photo-oxidation led to a slower turnover rate. A slow and probably nonspecific reaction finally impaired phosphorylation by ATP (not shown). Table II shows the steady-state values obtained for phosphorylation with [-³²P]ATP and [³²P]P_i. For comparison, the effects elicited by fluorescein are also shown. Although the E₁·Ca₂ form remained fully phosphorylatable by ATP, phosphorylation of Ca²⁺-free enzyme by orthophosphate was gradually impaired, showing that reactions attributed to the E₂ conformation of the enzyme were affected dramatically. The same results were obtained regardless of whether the pre-incubation with erythrosin was done in the presence or the absence of Ca²⁺. Therefore, the site where photo-inhibition takes place seems to be accessible to erythrosin in both the E₁ and E₂ conformations. As also shown in Table II, the effects observed with erythrosin were similarly obtained with fluorescein, in spite of fluorescein being a less effective photo-inhibitor and consequently requiring much higher dye concentrations. It is also worth noting that these results are totally different from those obtained with Ca²⁺-ATPase after labeling with FITC, where phosphorylation by ATP is drastically impaired (because the putative ATP binding site is occupied by the covalently bound label) and phosphorylation by P_i is not affected at all (Pick and Bassilian, 1981).

Table II. Steady-state levels of phosphoenzyme formed with ATP or Pi by the sarcoplasmic reticulum Ca2+-ATPase photo-oxidized with erythrosin or fluorescein The enzyme was phosphorylated after preincubation with the inhibitors for 10 min under room light. The values presented are the means of seven experiments ± S.E. [Dye] E-P with ATP E-P with Pi µM % Erythrosin 0.2 102.2  ± 1.9 94.6  ± 1.8 0.5 98.5  ± 5.0 97.2  ± 4.8 2.0 101.3  ± 4.2 69.2  ± 8.0 5.0 97.0  ± 6.1 58.9  ± 4.8 Fluorescein 10 101.7  ± 2.0 96.5  ± 1.5 20 98.9  ± 1.0 81.0  ± 5.4 100 94.3  ± 5.7 66.8  ± 12.0 200 86.8  ± 1.5 40.5  ± 2.0

As described above, the steady-state levels of ATP-derived phosphoenzyme, as well as the apparent affinity of the catalytic site for ATP, were not modified by photo-inhibition with erythrosin, although overall enzyme activity was markedly decreased. The time course for phosphorylation of Ca²⁺-ATPase by ATP is depicted in Fig. 4. In Fig. 4, A and B, control and photo-oxidized enzyme were preincubated in the presence of Ca²⁺ and Mg²⁺, and the phosphorylation reaction was started by the addition of ATP, whereas in Fig. 4, C and D, enzyme phosphorylation was initiated by the addition of ATP and Ca²⁺ to the enzyme previously exposed to light in the presence of EGTA and Mg²⁺ with or without the dye. In both cases, a marked reduction of the phosphorylation rate was observed in the photo-inhibited enzyme. However, inhibition with the dye induced a proportionally greater decrease (k  145 s¹ to k  23 s¹) in the initial apparent phosphorylation rate when the phosphorylation reactions were started with the Ca²⁺-bound enzyme (Fig. 4, A and B) than when enzyme was preincubated in EGTA (k  50 s¹, k  18 s¹) beforehand (Fig. 4, C and D). Conversely, maximal phosphorylation levels similar to control tended to be reached both by the Ca²⁺-incubated and EGTA-incubated enzyme. It must be stressed that the reaction rates observed for phosphorylation of the photo-oxidized enzyme are very similar regardless of the initial (i.e. whether Ca²⁺ was bound or not) enzyme conformation. At the concentrations used in this work, Ca²⁺ and ATP binding to the E₁ form of the enzyme are expected to be very fast when compared with the E₂-E₁ conformational transition rate (Scofano et al., 1979). Thus it is assumed that isomerization from E₂ to E₁ would be the rate-limiting step for phosphorylation of the native Ca²⁺-ATPase when beginning with the E₂ form of the enzyme. However, after photo-oxidation this rate-limiting step seems to be superseded due to slowing of a subsequent step, which makes the overall phosphorylation reaction rates very slow, and similar irrespective of the initial conformer. Changing the rate of ATP binding would be expected to change the K_d for ATP and shift the K_m to higher values, a change that was not observed for the catalytic site of the photo-oxidized enzyme (see Table I). Thus, it is likely that the phosphoryl transfer reaction was affected by reaction with erythrosin.

Photo-oxidation had a pronounced effect on the dephosphorylation rates of ATP-formed phosphoenzyme. The dephosphorylation reaction was much slower than control both for the forward (EGTA-induced) and backward (ADP-sensitive) directions. The EGTA-induced phosphoenzyme decay showed a pronounced decrease in the observed rate constants for the photo-oxidized enzyme, decreasing from k  3.36 s¹ to k  1.1 s¹ (Fig. 5, A and B). The extent of inhibition was higher the higher the dye concentrations used (not shown). Although photo-inhibition severely impaired phosphoenzyme decay in both the forward and reverse directions, the inhibition was much more accentuated on the ADP-sensitive pathway, and the typical two-exponential, fast ADP-sensitive decay (k₁  183 s¹; k₂  20 s¹) became so inhibited that it could be simply fitted with a slow (k  0.75 s¹), single exponential decay (Fig. 5, C and D). Binding of ADP to the phosphoenzyme was probably not impaired by photo-oxidation, because in these experiments the enzyme was already phosphorylated by ATP (evidencing nucleotide binding) and also because ADP was still able to protect against the well described glutaraldehyde-induced intramolecular cross-linking (McIntosh, 1992) in the same conditions and to the same extent for both the control and the photo-oxidized enzyme (Fig. 6A). Interestingly, erythrosin, in the absence of illumination, efficiently protected against this glutaraldehyde-induced cross-linking (Fig. 6B), suggesting that both ADP and erythrosin (and probably ATP also), may bind to a common site or induce similar enzyme conformations. Therefore, our results point toward the accumulation of a trapped, ADP-insensitive phosphoenzyme. This could be the E₂-P·Ca₂ or the E₂-P form or a modified form of E₁-P·Ca₂, which binds ADP but can not efficiently resynthesize ATP due to the lack of one or more amino acids essential to the synthesis reaction.

As cited earlier in this text, [⁴⁵Ca]Ca²⁺ binding at equilibrium to a static, nonhydrolyzing enzyme showed the same levels for control and erythrosin photo-inhibited enzyme. If the accumulated phosphoenzyme generated upon ATP phosphorylation were a Ca²⁺-free E₂-P form, one would expect a decrease in the total bound [⁴⁵Ca]Ca²⁺ and also in the ratio between bound Ca²⁺ and phosphoenzyme formed. This indeed was not the case, because in all the conditions tested, with varying degrees of inhibition, the ratio of Ca²⁺ bound to the phosphoenzyme formed was always around 2. This ratio was maintained even when ATPase activity was reduced to less than 10% of the control and even when the levels of phosphorylation with ATP were already partially impaired by photo-oxidation (Table III). Therefore, a possible accumulation of a Ca²⁺-free phosphoenzyme can be discarded, and the observed phosphoenzyme must thus be a Ca²⁺-bound intermediate.

Table III. Steady-state [45Ca]Ca2+ binding by the erythrosin photo-inhibited Ca2+-ATPase and correlation with the amount of phosphoenzyme formed with [-32P]ATP The maximum values obtained from three experiments were 8.4 ± 0.3 nmol/mg for occluded Ca2+ and 4.01 ± 0.2 nmol/mg for phosphoenzyme (mean ± S.E.). Occluded 45Ca2+ Phosphoenzyme % Control 100.0 100.0 2 µM erythrosin 93.0  ± 5.0 94.2  ± 0.5 5 µM erythrosin 80.2  ± 4.4 88.1  ± 2.8

TNP-ATP, a fluorescent ATP derivative that binds strongly but noncovalently to Ca²⁺-ATPase, was used to distinguish among the possible Ca²⁺ bound, phosphorylated enzyme forms. TNP-ATP fluorescence is enhanced upon binding to the enzyme, and fluorescence is further enhanced when phosphorylation by ATP or P_i occurs. This phenomenon, classically known as superfluorescence, is attributed to the accumulation of E₂-P·Ca₂ and E₂-P forms (Berman, 1986; Bishop et al., 1987). Binding of TNP-ATP to the Ca²⁺-ATPase was not disabled by the photo-inhibition nor by the remaining free erythrosin, because the addition of 2 µM TNP-ATP to both control and modified enzyme caused a fluorescence enhancement much greater than that observed following addition of the probe to enzyme-free solutions and because the fluorescence of the bound TNP-ATP was reduced to the same extent by the addition of ATP in the absence of Ca²⁺ with both control and photo-inhibited enzyme (not shown). The affinity for TNP-ATP (or possibly the conformation of its binding site) appeared to be modified, because the enhancement of TNP-ATP fluorescence upon binding to the ATPase was consistently slightly higher for the control than for the photo-oxidized enzyme (not shown). The superfluorescence of the enzyme-bound TNP-ATP was entirely suppressed when an 80% phosphorylatable but less than 10% catalytically competent photo-oxidized Ca²⁺-ATPase was phosphorylated with ATP (Fig. 7), showing that perhaps a hydrophilic/hydrophobic transition in the TNP-ATP binding site, linked to the transition from E₁-P·Ca₂ to E₂-P^·Ca₂ and responsible for the superfluorescence, was impaired. Indeed, the addition of ATP to the photo-oxidized enzyme (Fig. 7, right panel) induced a small decrease in fluorescence that could be due to binding of the nucleotide and accumulation of E₁-P·Ca₂, because (as cited above) the superfluorescence is observed when the accumulation of E₂-P·Ca₂ or E₂-P occurs.

DISCUSSION

The purpose of this study was to correlate the modification of amino acids of the SR Ca²⁺-ATPase by a dye that behaves as a nonhydrolyzable substrate analog with the effects observed on the different steps of substrate hydrolysis. This was achieved using the halogenated fluorescein derivative erythrosin B, which can induce photo-inhibition of the Ca²⁺-ATPase in a reaction that has been described as oxidizing residues located in particular environments, possibly within putative nucleotide binding site(s) (Lundblad and Noyes, 1984; Ray and Koshland, 1962).

Photo-oxidation with methylene blue (Yu et al., 1967) or rose bengal (Yu et al., 1974; Coffey et al., 1975) has been used to investigate ATP hydrolysis and Ca²⁺ transport by the Ca²⁺-ATPase, and early findings led to the conclusion that histidyl residues, which in these cases were shown to be the main target, are critical for Ca²⁺ binding and participate in ATP hydrolysis and phosphoenzyme formation and turnover. Some xanthene dyes are structurally similar to adenine nucleotides and have been shown to bind to several nonmembranous enzymes that have nucleotide binding sites, such as lactate dehydrogenase (Wassarman and Lentz, 1971), aspartate transcarbamylase (Jacobsberg et al., 1975), hexokinase (Yip and Rudolph, 1976), and creatine kinase (Sommerville and Quiocho, 1977). They also bind to membraneous ion-transporting enzymes, including shark rectal gland Na⁺,K⁺-ATPase (Skou and Esmann, 1981; Skou and Esmann, 1983), pig stomach H⁺,K⁺-ATPase (Helmich-de Jong et al., 1986), mitochondrial F₁ ATPase (Neslund et al., 1984), erythrocyte Ca²⁺-ATPase (Mugica et al., 1984; Gatto and Milanick, 1993), plant plasma membrane Ca²⁺-ATPase (De Michelis et al., 1993), yeast plasma membrane H⁺-ATPase (Wach and Gräber, 1991), and SR Ca²⁺-ATPase (Coffey et al., 1975; Morris et al., 1982; Murphy, 1988). For most of these ATPases, xanthene dyes were shown to reversibly inhibit ATP hydrolysis in the micromolar range, provided that photo-oxidation was avoided. The structural similarity of erythrosin with adenine nucleotides is well discussed in Neslund et al. (1984). Therefore, it is quite possible that fluorescein and its derivatives are binding to nucleotide sites of these enzymes. The nucleotide site or sites in different P-type ion-transporting ATPases are likely to be very similar in structure, as evidenced by amino acid homologies within P-ATPases, which reveal a highly conserved glycine-rich fragment in the segments that are predicted to form the ATP catalytic site (Taylor and Green, 1989).

A reagent frequently used for the study of ion-transporting pumps is the fluorescein derivative FITC, which in most cases binds covalently and stoichiometrically to a lysine residue in the putative nucleotide binding site, inducing a concomitant loss of the ability to bind ATP (Pick, 1981). Murphy (1988) demonstrated that Lys-515, which is specifically labeled in SR Ca²⁺-ATPase (Maruyama et al., 1989), is not unusually reactive toward FITC, indicating that attachment of the affinity label must be directed by the fluorescein moiety of the compound. However, Murphy's data and our own results show that the Ca²⁺-ATPase affinity for free fluorescein is not particularly high, being strongly increased (at least two orders of magnitude) by the addition of halogenated substitutes to the molecule. Many authors have used eosin isothiocyanate (Papp et al., 1987; Munkonge et al., 1988) and erythrosin isothiocyanate (Papp et al., 1987; Birmachu and Thomas, 1990; Voss et al., 1991) rather than FITC, due to their higher affinity and particular fluorescence characteristics, to study rotational movements of the SR Ca²⁺-ATPase. These authors concluded that both compounds bind to the same site and react with the same lysyl residue as FITC (Papp et al., 1987; Birmachu and Thomas, 1990). Consequently, it is expected that erythrosin itself must bind noncovalently and with high affinity to a nucleotide binding cleft. Furthermore, as the oxygen species generated upon excitation of the dye with light is rapidly deactivated in aqueous solutions (overall rate constant 5 × 10⁵ M¹. s¹ at pH 7.0, Halliwell and Gutteridge, 1989), the free radical must be produced very near the target modified amino acid. Thus, our reasoning is that erythrosin exerts photo-inhibition by modification of some residue(s) within the nucleotide binding site(s) of the SR Ca²⁺-ATPase, through a reaction that depends on the generation of singlet oxygen reactive species inside the site(s) by the light-excited, noncovalently bound dye (Halliwell and Gutteridge, 1989).

Yu et al. (1974) demonstrated that photo-inhibition of SR Ca²⁺-ATPase by rose bengal was due to the modification of histidyl residues, which were preferentially lost upon photo-oxidation, and not to peroxidation of phospholipids, consistent with Martonosi et al. (1972). However, Yu et al. (1974) reported that Ca²⁺ transport was more sensitive than ATPase activity to photo-inhibition with rose bengal. Morris et al. (1982) found a similar behavior using erythrosin but assumed that no photo-oxidation occurred in their experiments. We find that net Ca²⁺ transport is indeed inhibited more effectively than ATPase activity, but this is due to a Ca²⁺ leakage induced after some minutes of incubation with erythrosin, even in the absence of light.² Therefore, it is not clear whether the apparently higher sensitivity of Ca²⁺ transport to halogenated dyes is due to uncoupling of the enzyme by photo-oxidation of amino acids directly related to Ca²⁺ transport or to membrane permeabilization.

Our results show that the events related to phosphoryl transfer to and from the Ca²⁺-ATPase are drastically affected by photo-oxidation. Coffey et al. (1974) had already observed that both phosphorylation and dephosphorylation rates were impaired by photo-oxidation of SR vesicles. It does not seem that the residue(s) modified in our work, are directly involved in either Ca²⁺ binding (Coan and DiCarlo, 1990) or substrate binding (Lacapère et al., 1990), because neither the Ca²⁺ dependence of ATP hydrolysis nor the K_m1 for nucleotide hydrolysis is impaired by photo-oxidation. This is further supported by the fact that photo-oxidation also impairs phosphorylation by P_i, which does not depend on Ca²⁺ binding and does not need a tight fit to a nucleotide cleft.

The time course for phosphorylation of the photo-oxidized Ca²⁺-ATPase by ATP is adequately simulated by a model in which only the phosphoryl transfer and the rate constants related to the E₂-E₁ transition are changed. These simulations were based on the hydrolytic cycle of the Ca²⁺-ATPase presented in Scheme I, as originally proposed by Carvalho et al. (1976) and do not distinguish transitional subconformations, such as the sequential binding of Ca²⁺ ions (Inesi, 1987), and intermediate steps after nucleotide binding and before the phosphoryl transfer reactions (Stahl and Jencks, 1987; Petithory and Jencks, 1988). Most of the rate constants used in Table IV for the unmodified Ca²⁺-ATPase are based on other publications (see Table IV), and after small adjustments are adequate for the simulation of the curves shown in this work (see Figs. 4 and 5).

Table IV. Rate constants for the reactions catalyzed by native and photo-oxidized Ca2+-ATPase The constants presented here are apparent values that apply to the experimental conditions used in this work (pH 7.4, 25 °C, 80 mM KCl, 5 mM MgCl2, 0.05 mM CaCl2, and 15 µM ATP). k+n and kn are the rate constants calculated for the forward and reverse reactions in the absence of erythrosin. k+n and kn are the rate constants after photo-oxidation with 5 µM erythrosin. The rate constants omitted are assumed not to be modified by photo-oxidation. Step Reaction k+n kn k+n kn 1 E1 + 2Ca2+  E1 · Ca2 1 × 1013M2 s1a,b 10 s1a,b 2 E1 · Ca2 + ATP  E1 · Ca2 · ATP 2 × 107 M1 s1c 40 s1b,d,e 3 E1 · Ca2 · ATP  E1 P · Ca2 · ADP 240 s1d,f 400 s1f 20 s1 1 s1 4 E1 P · Ca2 · ADP  E1 P · Ca2 + ADP 3 × 103 s1b 1 × 107 M1 s1b 5 E1 P · Ca2  E2 P · Ca2 20 s1g 100 s1h 1 s1 20 s1 6 E2 P · Ca2  E2 P + 2Ca2+ 25 s1d 6.2 × 106 M1 s1a,d 7 E2 P  E2 · Pi 60 s1i,j 200 s1h 20 s1 10 s1 8 E2 · Pi  E2 100 s1j 1 × 104 M1 s1j 9 E2  E1 40 s1h 53 s1h 4 s1 2.6 s1 a  Pickart and Jencks, 1984. b  Alonso and Hecht, 1990 (with modifications from Teruel et al., 1987). c  Petithory and Jencks, 1988. d  Fujimori and Jencks, 1988. e  Pickart and Jencks, 1982. f  Nakamura et al., 1986. g  Inesi et al., 1982. h  This work. i  Stahl and Jencks, 1987. j  Inesi and de Meis, 1989.

All of the results obtained with the photo-oxidized Ca²⁺-ATPase presented in Figs. 4 and 5 can be reasonably simulated by assuming that the rate constants related to the phosphoryl transfer reactions (steps 3 and 7 in Scheme I) and to the interconversions between the E₁-P and E₂-P and E₂ and E₁ (steps 5 and 9 on Scheme I) are lower than in the control (Table IV). These values also predict the steady-state ATPase activity for the photo-oxidized enzyme at 15% of the control values, which is in good agreement with the observed experimental values (15-20%, see Fig. 1). The values for the constants after photo-oxidation were obtained by considering: (a) the low phosphorylation rate of E₁·Ca₂ by ATP (k₃); (b) the very slow dephosphorylation induced by ADP and EGTA (k₃); (c) the accumulation of a Ca²⁺-bound phosphoenzyme together with the observed loss of the superfluorescence of TNP-ATP (k₅ and k₅); and (d) the inhibition of phosphorylation by P_i (k₇ and k₇). The values of k₉ and k₉ were adjusted so as to account for the differences in phosphorylation rates between the Ca²⁺-bound enzyme and the Ca²⁺-depleted enzyme (estimated as 25% of the enzyme initially in the E₂ conformation).

There are several ways to explain the results obtained with the photo-oxidized Ca²⁺-ATPase. One or more of the oxidized residues could be involved in: (a) the coordination of the phosphate group to be transferred, (b) the binding and transfer of water molecules within the catalytic site, and (c) the nucleophilic attack of the phosphoryl and/or aspartyl group(s), which is the basis for phosphoryl transfer or hydrolysis. Taken together with results obtained elsewhere using halogenated photo-oxidants, our data are consistent with the notion that one or more amino acids susceptible to photo-oxidation by xanthene dyes is involved in substrate hydrolysis in the catalytic site of some, if not all, P-type ATPases.