Isolation of the Sarcoplasmic Reticulum Ca2+-ATPase from Rabbit Fast-Twitch Muscle

The sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) is a membrane protein that is destabilized during purification in the absence of calcium ions. The disaccharide trehalose is a protein stabilizer that accumulates in the yeast cytoplasm when under stress. In the present work, SERCA was purified by including trehalose in the purification protocol. The purified SERCA showed high protein purity (~95%) and ATPase activity. ATP hydrolysis was dependent on the presence of Ca2+ and the enzyme kinetics showed a hyperbolic dependence on ATP (Km = 12.16 ± 2.25 μM ATP). FITC labeling showed the integrity of the ATP-binding site and the identity of the isolated enzyme as a P-type ATPase. Circular dichroism (CD) spectral changes at a wavelength of 225 nm were observed upon titration with ATP, indicating α-helical rearrangements in the nucleotide-binding domain (N-domain), which correlated with ATP affinity (Km). The presence of Ca2+ did not affect FITC labeling or the ATP-mediated structural changes at the N-domain. The use of trehalose in the SERCA purification protocol stabilized the enzyme. The isolated SERCA appears to be suitable for structural and ligand binding studies, e.g., for testing newly designed or natural inhibitors. The use of trehalose is recommended for the isolation of unstable enzymes.

The isolation of membrane proteins/enzymes while preserving their native structure is of paramount importance for in vitro functional-structural experiments.In this Methods Protoc.2023, 6, 102 2 of 12 context, the isolation of a P-ATPase in high purity and catalytically active form can help to test interacting molecules identified by molecular docking and molecular dynamics simulations [15,[24][25][26].P-type ATPases are known to be remarkably labile to harsh media conditions; e.g., Ca 2+ -ATPase is known to be sensitive to high centrifugation speeds (i.e., high pressure), the presence of oxidative compounds, a relatively extreme pH and mild heat [27][28][29][30].Recently, the yeast plasma membrane H + -ATPase was isolated in its active hexameric state and at high purity [31].The existence of the H + -ATPase hexamer as a functional-structural state was later confirmed and its three-dimensional (3D) structure determined using cryo-electron microscopy [23].The isolation of the H + -ATPase hexamer was achieved using the disaccharide trehalose [31], a well-known protein stabilizer [32,33] that prevents protein unfolding and monomer dissociation [34,35].
In this work, SERCA was isolated from fast-twitch muscle using a purification protocol based on that used to isolate yeast plasma membrane H + -ATPase [31,36].The purified SERCA was of high purity and exhibited enzyme kinetics and other biochemical properties similar to those reported in the literature.Fluorescence labeling with FITC was used to test the integrity of the SERCA binding site and the identity of the enzyme as a P-type ATPase [37].The secondary structure of SERCA and the effect of ATP binding were analyzed using circular dichroism (CD) spectroscopy.The results showed that the isolated SERCA is suitable for functional and structural experiments, e.g., to test inhibitors (naturally occurring or designed and synthesized in laboratories) identified by molecular docking and molecular dynamics simulation [15,[24][25][26].

Sarcoplasmic Reticulum Isolation
Muscle tissue was obtained from wild-type Oryctolagus cuniculus from a local animal handling unit (INE/CITES/DGVS-ZOO-E0055-SLP-98).Sarcoplasmic reticulum vesicles (SRVs) were prepared as described by Champeil et al. (1978) [38], with slight modifications [26].Briefly, fast-twitch muscles were macerated in a Oster Pro blender (Sunbeam Products Inc., Miami, FL, USA) in three volumes of 100 mM KCl, with 1 min on and 1 min in ice-cold rest repeated three times.Tissue debris was removed using centrifugation (1532× g) at 4 • C for 5 min; a second centrifugation (4256× g) was performed if necessary.The supernatant was collected and homogenized using a tissue grinder and WiseStir HS-30E (Daihan Scientific Co., Seoul, Republic of Korea), then centrifuged (10,048× g) at 4 • C for 15 min.The resulting supernatant containing sarcoplasmic reticulum was centrifuged (33,110× g) at 4 • C for 120 min.The pellets were suspended in 0.5 M sucrose and centrifuged (12,111× g) at 4 • C for 15 min.The supernatant was diluted to 0.6 M KCl and 0.15 M sucrose and centrifuged (34,310× g) at 4 • C for 165 min.The SRVs sheets were suspended in 0.3 M sucrose, 0.1 M KCl, and 5 mM Tris-HCl, pH 7.0.Protein concentration was determined using the Lowry assay [39,40], using human serum albumin as the protein standard.The SRVs (30-32 mg/mL) were aliquoted in 1 mL volumes and stored at −72 • C until use.

Enzyme Kinetics
ATPase activity was measured at 37 • C using an enzyme-coupled assay, as previously described [41,42].The reaction mixture (1 mL) consisted of 50 mM MOPS (pH 7.0), 1 mM EGTA, 80 mM KCl, 5 mM MgCl 2 , 3 mM CaCl 2 , 5 mM phosphoenolpyruvate, 250 µM NADH, and ATP concentration as indicated (0.001 to 0.25 mM).The reaction solution was homogenized carefully by vortexing and then incubated at 37 • C for 10 min.Then, 0.9 U L-lactate dehydrogenase (LDH) and 1.5 U pyruvate kinase (PK) were added and the ATPase reaction was initiated by the addition of 10 µg of the purified SERCA.The NADH formed was monitored by the change in absorbance intensity at a wavelength (λ) of 340 nm using an 8453UV/VIS spectrophotometer (Agilent Technologies, Waldbronn.DE) equipped with a thermostatted cell holder.The rate of ATP hydrolysis was calculated from the slope of the linear portion of each curve using the molar extinction coefficient (ε) of NADH (λ = 6220 M −1 •cm −1 ).Velocity data were fitted to the Michaelis-Menten Equation (1) (Equation ( 1)) using non-linear regression with the Origin 6.0 software: where v is the velocity, V max is the maximum velocity, [S] is the ATP concentration and K m is the Michaelis-Menten constant.

SERCA Labeling with Fluorescein Isothiocyanate (FITC)
Covalent labeling of SERCA with FITC was performed as previously described with slight modifications [43][44][45].Briefly, purified SERCA (20 µg) was suspended in 50 µL (final volume) labeling buffer (100 mM KCl, 5 mM MgCl 2 , and 30 mM Tris-HCl, pH 8.9) containing 1 mM FITC.After mixing using vortexing, the samples were incubated for different times (15, 10, 7, 5, and 2 min) in the dark at room temperature, and labeling reaction was stopped with 1 volume of ice-cold stopping buffer (480 mM sucrose and 48 mM MOPS, pH 7.0), containing ATP (5 mM); the mixture was incubated on ice for 5 min in the dark.FITC-labeled SERCA was then subjected to SDS-PAGE.The clear gels were exposed to UV light (λ = 302 nm) and photodocumented using a benchtop UV transilluminator (Cole-Parmer, Vernor Hills, IL, USA) and a Coolpix B500 camera (Nikon Corp., Tokyo, Japan).After photographic documentation, the gel was stained with Coomassie blue.

Circular Dichroism (CD) Spectrum of SERCA
Purified SERCA (3 µM) was suspended in 400 µL of 10 mM phosphate buffer (pH 7.0) at 25 • C. Aliquots of ATP were added stepwise as indicated (5-100 µM final concentration) in the absence and presence of Ca 2+ (200 µM).Far-UV spectra (λ range of 190-260 nm) were recorded at 50 nm/min in a Jasco J1500 spectropolarimeter (Jasco Inc., Tokyo, Japan) using a 0.1 cm path length cell at 25 • C. The internal resolution of the data was 1 nm and the bandwidth was 1 nm.
In this work, the purification of rabbit fast-twitch-muscle SERCA was carried out using the purification methodology used for the isolation of yeast plasma membrane H + -ATPase, as it allows the successful isolation of the functional hexameric state [31].Sarcoplasmic reticulum vesicles (SRVs) were obtained, and the kinetic parameters (K m and V max ) for ATPase activity of the isolated SRVs were similar to those reported in the literature (V max = 7.0 µmoles ATP/min•mg protein and K m = 15.1 µM ATP) [26].The purification of SERCA from SRVs was then performed as described in the Methods [36]; SRVs (SERCA ~110 kDa) at different steps of isolation were monitored using SDS-PAGE (Figure 1A).SRVs were solubilized with detergent and importantly, after centrifugation (103,000× g) on a trehalose concentration gradient, a clear pellet was observed at the bottom of the centrifuge tubes containing SERCA in high purity (Figure 1B).Samples at different tube heights were also taken and analyzed.Notably, SERCA was only observed in the pellet (Figure 1B).The purity of SERCA was ≈95%, as determined using densitometry [31].Importantly, when SERCA was ultracentrifuged in the absence of trehalose, it denatured, forming white insoluble aggregates and irreversibly losing ATPase activity (Figure 1B, last lane).The isolation protocol was tested at least three times with similar results for protein purity.Suspended SERCA was stored at −71 • C until use.

FITC Labeling of SERCA
In SERCA, FITC-labeling indicates the intactness and interacting functionality of the ATP-binding site [26,37,43,44].FITC interacts with the nucleotide binding site of the enzyme [37], resulting in the formation of a covalent bond between FITC and a lysine (Lys) residue [37,45,69,70].SERCA was subjected to FITC labeling at different incubation times as described in the Methods, followed by SDS-PAGE and photo-documentation using UV irradiation (Figure 3A).The gel in Figure 3A shows a single bright green fluorescent band corresponding to the molecular weight of SERCA (MW ~110 kDa) (Figure 3A).The gel was then stained with Coomassie blue (Figure 3B).The resulting blue bands (~110 kDa) were superimposed on those showing green fluorescence under UV irradiation (Figure 3A).FITC labeling was also performed in the presence of 3 mM Ca 2+ , but no change in FITC labeling was observed.FITC labeling occurred relatively rapidly, as no significant differences were observed at the incubation times tested (Figure 3).

FITC Labeling of SERCA
In SERCA, FITC-labeling indicates the intactness and interacting functionality of the ATP-binding site [26,37,43,44].FITC interacts with the nucleotide binding site of the enzyme [37], resulting in the formation of a covalent bond between FITC and a lysine (Lys) residue [37,45,69,70].SERCA was subjected to FITC labeling at different incubation times as described in the Methods, followed by SDS-PAGE and photo-documentation using UV irradiation (Figure 3A).The gel in Figure 3A shows a single bright green fluorescent band corresponding to the molecular weight of SERCA (MW ~110 kDa) (Figure 3A).The gel was then stained with Coomassie blue (Figure 3B).The resulting blue bands (~110 kDa) were superimposed on those showing green fluorescence under UV irradiation (Figure 3A).FITC labeling was also performed in the presence of 3 mM Ca 2+ , but no change in FITC labeling was observed.FITC labeling occurred relatively rapidly, as no significant differences were observed at the incubation times tested (Figure 3).

Secondary Structure of SERCA
The secondary structure of SERCA was analyzed using circular dichroism (CD) spectroscopy.SERCA (3 µM) was suspended in 10 mM phosphate buffer (pH 7.0), and the circular dichroism (CD) spectrum was recorded at 25 °C (Figure 4A).ATP was then added stepwise (0-100 µM) and CD spectra were recorded after each addition.The results showed a slight change in molar ellipticity as a function of ATP concentration at a wavelength (λ) of 225 nm (Figure 4A), but not at λ of 212 nm.A hyperbolic pattern was observed at ATP saturation (Figure 4B), which was similar to that observed in a recombinant engineered Ca 2+ -ATPase N-domain [37].Notably, the secondary structural changes in α-helices mediated by the presence of ATP occurred in the µM region for the ATP affinity determined by enzyme kinetics (Figure 2).Therefore, the change in CD intensity at λ of 225 nm appears to correspond to changes in secondary structure (α-helices) induced by ATP binding [71].The experiment was repeated in the presence of 200 µM Ca 2+ , but no significant

Secondary Structure of SERCA
The secondary structure of SERCA was analyzed using circular dichroism (CD) spectroscopy.SERCA (3 µM) was suspended in 10 mM phosphate buffer (pH 7.0), and the circular dichroism (CD) spectrum was recorded at 25 • C (Figure 4A).ATP was then added stepwise (0-100 µM) and CD spectra were recorded after each addition.The results showed a slight change in molar ellipticity as a function of ATP concentration at a wavelength (λ) of 225 nm (Figure 4A), but not at λ of 212 nm.A hyperbolic pattern was observed at ATP saturation (Figure 4B), which was similar to that observed in a recombinant engineered Ca 2+ -ATPase N-domain [37].Notably, the secondary structural changes in α-helices mediated by the presence of ATP occurred in the µM region for the ATP affinity determined by enzyme kinetics (Figure 2).Therefore, the change in CD intensity at λ of 225 nm appears to correspond to changes in secondary structure (α-helices) induced by ATP binding [71].The experiment was repeated in the presence of 200 µM Ca 2+ , but no significant differences in the CD spectra (200-260 nm) were observed, in agreement with that reported by Csermely et al. (1987), and by Shivanna and Rowe (1997) [58,71].

Discussion
Most integral membrane proteins/enzymes are difficult to isolate in their native functional state due to their hydrophobic nature [72].The detergent solubilization of biological membranes usually leads to the disruption of embedded macromolecular complexes, dissociation of protein/enzyme oligomers, protein unfolding, and consequently the loss of function/activity [58].Any effective method of isolating membrane proteins must minimize this disruption of the delicate structural arrangement.
The use or inclusion of cosmotropic agents in isolation buffers can help to improve the retention of the native structure and, thus, the function of proteins [30,73,74].In this sense, the disaccharide trehalose is a well-known protein stabilizer synthesized by yeast, plants, and other organisms under harsh conditions such as heat shock temperatures, desiccation, and others [75][76][77][78][79][80][81][82][83][84][85][86][87].Various studies have shown that trehalose is superior in protein stabilization when compared to other saccharides and polyols [32,33,88,89].The molecular mechanism by which trehalose stabilizes and protects the three-dimensional structure of proteins is essentially well understood [33,34,88,[90][91][92].With regard to P-ATPases, the disaccharide trehalose has been shown to be able to stabilize H + -ATPase under various incubation/storage conditions (mainly dehydration and heat) [32,33], but also during the H + -ATPase purification process [31], i.e., the presence of trehalose prevents the loss of the native structure during H + -ATPase isolation, thus allowing macromolecular complexes formed by the enzyme hexamer to be isolated [31].The oligomeric state of the H + -ATPase is known to be highly labile.In plants, the H + -ATPase hexamer is stabilized by the fungal toxin fusicoccin via 14-3-3 protein binding [93].In yeast, trehalose appears to prevent the dissociation of the H + -ATPase hexamer, thus avoiding the loss of its quaternary structure and ATPase activity [31,33].Notably, the 3D structure of the Ca 2+ -ATPase in its dimeric state is currently lacking; similarly, the oligomeric structure of other P-type ATPases remains to be determined.
In this work, SERCA was purified in both a relatively high yield and purity by incorporating trehalose as a protein stabilizer.Trehalose is useful in preserving both structure and function, as evidenced by results obtained from FITC labeling, secondary structure

Discussion
Most integral membrane proteins/enzymes are difficult to isolate in their native functional state due to their hydrophobic nature [72].The detergent solubilization of biological membranes usually leads to the disruption of embedded macromolecular complexes, dissociation of protein/enzyme oligomers, protein unfolding, and consequently the loss of function/activity [58].Any effective method of isolating membrane proteins must minimize this disruption of the delicate structural arrangement.
The use or inclusion of cosmotropic agents in isolation buffers can help to improve the retention of the native structure and, thus, the function of proteins [30,73,74].In this sense, the disaccharide trehalose is a well-known protein stabilizer synthesized by yeast, plants, and other organisms under harsh conditions such as heat shock temperatures, desiccation, and others [75][76][77][78][79][80][81][82][83][84][85][86][87].Various studies have shown that trehalose is superior in protein stabilization when compared to other saccharides and polyols [32,33,88,89].The molecular mechanism by which trehalose stabilizes and protects the three-dimensional structure of proteins is essentially well understood [33,34,88,[90][91][92].With regard to P-ATPases, the disaccharide trehalose has been shown to be able to stabilize H + -ATPase under various incubation/storage conditions (mainly dehydration and heat) [32,33], but also during the H + -ATPase purification process [31], i.e., the presence of trehalose prevents the loss of the native structure during H + -ATPase isolation, thus allowing macromolecular complexes formed by the enzyme hexamer to be isolated [31].The oligomeric state of the H + -ATPase is known to be highly labile.In plants, the H + -ATPase hexamer is stabilized by the fungal toxin fusicoccin via 14-3-3 protein binding [93].In yeast, trehalose appears to prevent the dissociation of the H + -ATPase hexamer, thus avoiding the loss of its quaternary structure and ATPase activity [31,33].Notably, the 3D structure of the Ca 2+ -ATPase in its dimeric state is currently lacking; similarly, the oligomeric structure of other P-type ATPases remains to be determined.
In this work, SERCA was purified in both a relatively high yield and purity by incorporating trehalose as a protein stabilizer.Trehalose is useful in preserving both structure and function, as evidenced by results obtained from FITC labeling, secondary structure analysis (CD spectrum), and ATPase activity.This protocol offers advantages when compared to published molecular biology protocols; e.g., it does not require additional reagents, material, and processing enzymes (for gene cloning and protein expression and purification) or equipment, and mainly, the protein purified is the native enzyme.In regard to its disadvantages, the presence of other native residual proteins may be a problem.Thus, the SERCA isolation method described here will allow future structural/functional/regulatory studies, for example (a) to understand of functional divergence of isozymes [94] (i.e., the functional role of amino acid sequence variation) and the regulation of activity [15]; (b) the structural-mechanistic defect in mutation-mediated diseases [95][96][97][98][99][100]; (c) to test new compounds with inhibitory properties in a given Ca 2+ -ATPase pump (e.g., cardiac) [101], i.e., the identification of the structural target, and mechanism of inhibition of natural or newly designed inhibitors [101]; (d) the testing of molecular docking and molecular dynamics simulation results; (e) the clarification of the ion transport mechanism [48]; and (f) the role of specific structural dynamics in its function.The high isolation efficiency (~95%) of SERCA and PMA1 are examples of improving the effectiveness of the purification methodology developed in our laboratory [31,36].In addition, it is expected that the protocol will be applied to efforts to purify other P-ATPases and unstable proteins (cytosolic and integral membrane).

Figure 2 .
Figure 2. Enzyme kinetics of isolated SERCA.The assay for ATPase activity was performed at 37 • C using an enzyme−coupled assay (SERCA−PK−LDH) in buffer 50 mM MOPS, pH 7. (A) Dependence of SERCA specific activity (•) on ATP concentration.Data points represent the mean of three independent experiments plus or minus standard deviation.The rate of ATP hydrolysis showed a hyperbolic dependence on ATPase kinetics, so the data were fitted to the Michaelis-Menten equation (Equation (1)) using non−linear regression (V max = 1.68 ± 0.09 µmoles ATP/min•mg protein, and K m = 12.16 ± 2.25 µM ATP).(B) Lineweaver-Burk plot of the data in (A) (≤).

Figure 3 .
Figure 3. (A) FITC labeling of SERCA.Purified SERCA was incubated in the presence of FITC for different times and then subjected to SDS-PAGE; lanes: 1, 15 min; 2, 10 min; 3, 7 min; 4, 5 min; 5, 2 min.The clear gel was exposed to UV light (λ = 302 nm) and photo-documented.Free FITC is at the bottom of the gel.(B) Coomassie blue staining of the gel in (A).The lanes were loaded with 20 µg of SERCA.

Figure 3 .
Figure 3. (A) FITC labeling of SERCA.Purified SERCA was incubated in the presence of FITC for different times and then subjected to SDS-PAGE; lanes: 1, 15 min; 2, 10 min; 3, 7 min; 4, 5 min; 5, 2 min.The clear gel was exposed to UV light (λ = 302 nm) and photo-documented.Free FITC is at the bottom of the gel.(B) Coomassie blue staining of the gel in (A).The lanes were loaded with 20 µg of SERCA.

Figure 4 .
Figure 4. Effect of the presence of ATP on the circular dichroism (CD) spectrum of isolated SERCA.(A) CD spectra of SERCA at 25 °C.ATP was added stepwise to a SERCA suspension, and then mixed, and a CD spectrum was obtained (three accumulations); the assay was repeated three times, and a representative experiment is shown.(B) Effect of ATP on CD intensity at λ of 225 nm.Data show mean and standard deviation from experiments in (A).

Figure 4 .
Figure 4. Effect of the presence of ATP on the circular dichroism (CD) spectrum of isolated SERCA.(A) CD spectra of SERCA at 25 • C. ATP was added stepwise to a SERCA suspension, and then mixed, and a CD spectrum was obtained (three accumulations); the assay was repeated three times, and a representative experiment is shown.(B) Effect of ATP on CD intensity at λ of 225 nm.Data show mean and standard deviation from experiments in (A).