©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Reconstitution of Recombinant Phospholamban with Rabbit Skeletal Ca-ATPase (*)

Laxma G. Reddy (1), Larry R. Jones (2)(§), Steven E. Cala (2), Jeffrey J. O'Brian (3), Suren A. Tatulian (1), David L. Stokes (1)

From the (1) Department of Molecular Physiology and Biological Physics, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908, the (2) Krannert Institute of Cardiology, Department of Medicine, Indiana University School of Medicine, Indianapolis, Indiana 46202, and the (3) DuPont Merck Pharmaceutical Company, Wilmington, Delaware 19806

ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca-ATPase in response to -adrenergic stimulation. We have used the baculovirus expression system in Sf21 cells to express milligram quantities of wild-type PLB. After purification by antibody affinity chromatography, the function of this recombinant PLB was tested by reconstitution with Ca-ATPase purified from skeletal SR. The results obtained with recombinant PLB were indistinguishable from those obtained with purified, canine cardiac PLB. In particular, PLB reduced the apparent calcium affinity of Ca-ATPase but had no effect on V. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca-ATPase by 50%. This inhibition was fully reversed by addition of a monoclonal antibody to PLB, which mimics the physiological effects of PLB phosphorylation. Maximal PLB regulatory effects occurred at a molar stoichiometry of 3:1, PLB/Ca-ATPase. We also investigated peptides corresponding to the two main domains of PLB. The membrane-spanning domain, PLB, appeared to uncouple ATPase hydrolysis from calcium transport, even though the permeability of the reconstituted vesicles was not altered. The cytoplasmic peptide, PLB, had little effect, even at a 300:1 molar excess over Ca-ATPase.


INTRODUCTION

Phospholamban is a small polypeptide of 52 amino acid residues, which is composed of an N-terminal, cytoplasmic domain and a C-terminal, membrane-spanning domain (Fujii et al., 1987; Simmerman et al., 1986). In response to -adrenergic stimulation, PLB() regulates the rate of calcium pumping by the Ca-ATPase of cardiac SR (Colyer, 1993; Lindemann et al., 1983; Tada and Kadoma, 1989) and to a lesser extent of SR from slow-twitch (Briggs et al., 1992; Kirchberger and Tada, 1976) and smooth muscle (Raeymaekers et al., 1990). Although the mechanism is not entirely clear, PLB appears to interact with Ca-ATPase and thus suppresses calcium uptake into the SR; phosphorylation of PLB either by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase relieves this suppression (Colyer, 1993; Tada and Kadoma, 1989). PLB is not present in fast-twitch muscle fibers (Jones et al., 1985; Jorgensen and Jones, 1986; Kirchberger and Tada, 1976), but it has been shown to be capable of regulating the fast-twitch isoform of Ca-ATPase (SERCA1) in both reconstitution (Szymanska et al., 1990) and coexpression (Toyofuku et al., 1993) experiments.

To study the specific effects of PLB on the reaction cycle of Ca-ATPase, the enzyme has been reconstituted either with native PLB (Kim et al., 1990; Szymanska et al., 1990) or with synthetic peptides corresponding to domains of PLB (Kim et al., 1990; Sasaki et al., 1992). Unfortunately, results have not been conclusive. On the one hand, studies with synthetic peptides showed a reduction both in apparent calcium affinity of Ca-ATPase and in its maximal pumping rate ( V), measured at saturating calcium concentrations. In particular, the hydrophobic, membrane-spanning domain decreased the calcium affinity, and the hydrophilic, cytoplasmic domain decreased the V(Hughes et al., 1994; Sasaki et al., 1992). On the other hand, this same cytoplasmic domain has been reported by other groups to have no effect on calcium transport by native SR lacking PLB, either from skeletal muscle (Vorherr et al., 1992) or from atrial tumor cells (Jones and Field, 1993). The controversy is furthered by recent, thorough studies of cardiac SR indicating that PLB only affects calcium affinity and not V(Cantilina et al., 1993; Morris et al., 1991). Finally, PLB has been observed to form calcium-selective channels in planar lipid bilayers (Kovacs et al., 1988), suggesting an alternative, but not necessarily exclusive, mechanism for its action whereby PLB reduces calcium pumping rates by generating a calcium-selective leak across the SR membrane.

All of the reconstitution approaches reported so far suffer from two problems: the requirement of a large molar excess of PLB, or its fragments, relative to Ca-ATPase ( e.g. >100:1) to produce an effect and the small changes in enzyme activity achieved even with such high molar ratios. In such reconstituted systems, PLB inhibitory effects are generally much smaller than those commonly observed in intact cardiac SR vesicles containing native PLB. These problems could be due to suboptimal systems for reconstitution or perhaps to an undesirable effect of excess PLB on the properties of the lipid vesicles. Thus, we felt it important to develop a more efficient method of coreconstitution. In this work, we report on expression and purification of milligram quantities of PLB from Sf21 insect cells (Cala et al., 1993). A method is described for reconstitution of recombinant PLB at reasonably low stoichiometries to purified, skeletal Ca-ATPase (8:1, mol/mol), and we show that this recombinant PLB is competent to regulate Ca-ATPase. Using this method, we have addressed the mechanism of this regulation by measuring calcium uptake, ATPase activity, and the permeability of the vesicles to calcium. In addition, we have compared the effects of intact PLB with those of two peptides, corresponding to the membrane-spanning and cytoplasmic domains of PLB.


MATERIALS and METHODS

Preparation of Ca-ATPase Skeletal SR vesicles were prepared from the white muscle in the hind leg of rabbit by the method of Inesi and Eletr (1972). Ca-ATPase was purified from these SR vesicles by affinity chromatography using Reactive Red 120 as described previously (Stokes and Green, 1990). After eluting purified Ca-ATPase from the column in a buffer containing 1 m M CaCl, 1 m M MgCl, 20% glycerol, 20 m M MOPS (pH 7.0) and 0.1% CE, the peak fractions were pooled to give a protein concentration of 3-4 mg/ml at a yield of 25%. SDS-PAGE demonstrated that Ca-ATPase represented >98% of this purified protein with an ATPase activity of 8-10 µmol/mg/min at 25 °C; given this purity, the amount of calcium-independent ATPase activity, which often represents 5-10% of activity from SR, was negligible. The protein was stored at -80 °C after adding 1 mg of lipid/mg of protein and was found to retain full activity for 4-6 months under these conditions. Cardiac SR was prepared by the method of Jakab and Kranias (1988). Preparation of PLB

Construction of PLB Recombinant Baculovirus

cDNA encoding PLB was obtained as an EcoRI fragment from a canine cardiac gt10 library (Palmer et al., 1991) and ligated into pBluescript (Stratagene). This cDNA fragment contained the protein coding region for PLB, as well as 194 and 254 base pairs of 5`- and 3`-untranslated sequences, respectively. This cDNA was subcloned into the EcoRI cloning site of pVL1393. The transfer plasmid was cotransfected with wild-type baculovirus AcNPV using a calcium phosphate transfection kit (Invitrogen). Recombinant baculovirus clones were enriched by limiting dilution and screening infected cell lysates with a dot-blot method (Manns and Grosse, 1991) employing the PLB monoclonal antibody 2D12. Cell lysates, highly enriched in recombinant virus containing the PLB insert, were then used for plaque purification by standard methods (Levin and Richardson, 1990).

Expression of Recombinant PLB

For standard purification of PLB, 500 ml of Sf21 cells at a density of 1.5 10cells/ml were grown in suspension with PLB recombinant virus using a multiplicity of infection of 5-10. The cell suspension was incubated in a 4-liter Erlenmeyer flask in an orbital shaker for 2.5-3 days at 90 rpm and 27 °C in Grace's medium containing 10% fetal bovine serum, 0.1% Pluronic F68, 100 units/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B. At the end of the incubation period, the cells were sedimented and resuspended in 250 ml of 100 m M NaCO(pH 11.4) on ice, followed by centrifugation at 21,000 rpm for 30 min in a Beckman 21 rotor. The resulting carbonate-extracted pellets, enriched in PLB, were resuspended in 0.25 M sucrose and stored frozen at -20 °C.

Purification of Recombinant PLB

PLB was purified from carbonate-extracted pellets by monoclonal antibody affinity chromatography. For a typical purification, carbonate-extracted pellets from nineteen 500-ml infections were pooled yielding 1.53 g of protein, which was incubated at room temperature for 20 min in 590 ml of 17 m M MOPS, 1% SDS, and 3.5% Triton X-100 (pH 7.4). The suspension was then sedimented at 20,000 rpm for 20 min in a Beckman 21 rotor, and the supernatant, containing the detergent-solubilized PLB, was loaded over a 55-ml protein A-agarose (Sigma, P-1406) column with covalently attached PLB monoclonal antibody 2D12. Monoclonal antibody density on the protein A-agarose beads was 8 mg of antibody/ml of beads; covalent coupling of antibody to protein A was performed using dimethylpimelimidate (Harlow and Lane, 1988). The detergent-solubilized PLB was passed through the column two times at room temperature over a period of 2-3 h. The column was then eluted with seven consecutive 45-ml washes of 20 m M MOPS, 0.5 M NaCl, and 0.25% Zwittergent 3-14 (pH 7.2), followed by eight consecutive 45-ml washes with 20 m M MOPS, 1% -OG (pH 7.2). Purified PLB was then eluted with seven consecutive 45-ml washes of ice-cold 20 m M glycine, 1% -OG (pH 2.4). Each eluate with pH 2.4 buffer was recovered in 4.3 ml of 1 M MOPS to bring the final pH to 7.1. The first six fractions eluted with the glycine-containing buffer were then combined and concentrated to a final volume of 5.7 ml using a 50-ml Amicon and PM-10 membrane. Solid dithiothreitol (5 m M final concentration) was added to the pooled PLB fractions during Amicon concentration. The sample was then centrifuged at 100,000 rpm for 15 min in a TL 100.3 rotor to remove turbid material, which varied in amount from preparation to preparation but which did not contain PLB. The clear supernatant containing the purified PLB was stored in small aliquots at -40 °C. The protein concentration (Schaffner and Weissman, 1973) was 3.0 mg/ml, giving a final yield of 17.1 mg of purified PLB from 1.53 g of carbonate-extracted pellet protein.

Purification of canine cardiac PLB was done as described previously (Jones et al., 1985).

SDS-PAGE was used to document the purity of these PLB preparations and was performed according to Laemmli (1970) using a 7-18% gradient of polyacrylamide in the running gel and 3% polyacrylamide in the stacking gel. Preparation of PLB Fragments The cytoplasmic fragment of PLB, corresponding to residues 1-31, was synthesized by the solid-phase method in an Applied Biosystems 431A Peptide Synthesizer using 0.127 g of PAM Boc- L-leucine resin support at 0.79 mmol/g. The amino groups were protected by t-butoxycarbonyl groups, aspartic and glutamic acid side chains by benzyl esters, and arginine by Mts groups. After acetylating the N terminus by adding acetic acid (1 mmol to a final empty cartridge on the synthesizer), the peptide was cleaved from the resin with HF. After lyophilization, the peptide was purified by preparative, reverse-phase high performance liquid chromatography, eluting with a linear gradient starting with 0.1% trifluoroacetic acid and ending with 0.1% trifluoroacetic acid, 70% acetonitrile on a 5 25-cm C18 Vydak preparative column. The final purity of this peptide was verified by mass spectroscopy and by amino acid analysis (Table I).

The membrane-spanning domain of PLB was prepared by proteolysis of recombinant PLB, as described previously (Tatulian et al., 1995; Simmerman et al., 1986). Briefly, PLB was incubated for 24 h with trypsin at a weight ratio of 10:1 (PLB/trypsin) at room temperature. Several small peptides were generated from the cytoplasmic region of PLB by this method, leaving the membrane-spanning domain intact as a limit peptide (residues 26-52). After the passage of the sample through a small CM-Sepharose column to remove trypsin, the membrane-spanning domain was separated from the remaining smaller peptides by repeated centrifugation through a Centricon 30 filter (Amicon). The larger, hydrophobic region was retained by the membrane, while the smaller, water-soluble peptides were washed through the membrane. The final purity was determined by SDS-PAGE, as well as by protein sequencing, and has been documented in a previous publication (Tatulian et al., 1995). Reconstitution of Ca-ATPase with PLB and PLB Hydrophobic Peptide Two methods were used to reconstitute Ca-ATPase with PLB in lipid vesicles. The methods were similar except in the way that PLB was added. For method I, pure lipid vesicles were made by reverse-phase evaporation with a 10:1 weight ratio of EYPC to EYPA (Avanti Polar Lipids). Eight mg of these vesicles were completely solubilized with 16 mg of detergent (-OG, CE, or Triton X-100) in 10 m M imidazole (pH 7.0), 100 m M potassium oxalate in a final volume of 1.0 ml. To this mixture, 100 µg of purified Ca-ATPase and 42 µg of recombinant PLB were added such that the lipid-to-Ca-ATPase weight ratio was 80:1 and the Ca-ATPase-to-PLB molar ratio was 1:8. Reconstitution was accomplished by controlled removal of detergent with SM2 Biobeads (Bio-Rad): 320 mg in the case of -OG and CEand 640 mg in the case of Triton X-100. These solutions were stirred for 3 h at room temperature, and proteoliposomes were carefully removed from the Biobeads with a pipette (Levy et al., 1990a, 1990b, 1992).

For method II, 84 µg of recombinant PLB were lyophilized; then, a solution containing 16 mg of EYPC and EYPA (10:1 weight ratio) in 0.8 ml of CHClwas added, followed by 1 or 2 drops of trifluoroethanol to help bring PLB into solution. The solvents were evaporated under dry nitrogen gas, thus making a thin film of lipid, detergent, and PLB on the walls of the flask. After adding 1 ml of 10 m M imidazole (pH 7.0), the flask was vortexed vigorously to suspend the lipid/PLB mixture, which was then sonicated for 20-30 s (Branson Sonifier with a microtip) at 50% duty cycle to make unilamellar vesicles. Finally, 8 mg of these vesicles were added to 100 µg of purified Ca-ATPase and 16 mg of -OG in a solution containing 10 m M imidazole (pH 7.0) and 100 m M potassium oxalate. The detergent was then removed by incubation with 320 mg of Biobeads for 3 h at room temperature.

Proteoliposomes containing PLBwere prepared by incorporating 10 µg of peptide into 4 mg of EYPC/EYPA vesicles (10:1 weight ratio). To do this, these vesicles were first made by drying lipid into a thin film, by resuspending the lipid in 0.25 ml of 10 m M imidazole (pH 7.0), and then by sonicating for 1 min with a 50% duty cycle. After adding 10 µg of PLBin 0.2 mg of -OG, -OG was removed with 10 mg of Biobeads. Incorporation of purified Ca-ATPase into these peptide-containing proteoliposomes was done according to method II, described above, with a Ca-ATPase-to-peptide molar ratio of 1:8 and a lipid-to-Ca-ATPase weight ratio of 80:1.

Because PLBis a soluble peptide, it was not necessary to incorporate it into the vesicles. Rather, 60-300 µ M peptide was incubated with reconstituted proteoliposomes (containing 10 µ M Ca-ATPase) for 30 min at room temperature. After a 14-fold dilution into the calcium uptake assay mixture, assays for calcium uptake and ATPase activity were performed.

In all cases, control experiments involved omitting PLB, but adding the same amount of the solution in which PLB, or the peptide, was suspended. Assays of Calcium Uptake, ATPase Activity, and Vesicle Permeability Proteoliposomes were incubated with a monoclonal antibody to PLB (2D12, Briggs et al., 1992; Sham et al., 1991) on ice for 15 min prior to the calcium uptake assay such that the molar ratio of PLB to antibody was 1:1; control experiments included only the buffer in which the antibody was suspended. Ca uptake was measured at 25 °C by the microfiltration method (Martonosi and Feretos, 1964) in an assay volume of either 1.5 ml (method I) or 0.750 ml (method II). Each assay employed 2 µg of Ca-ATPase in a solution containing 50 m M imidazole (pH 7.0), 95 m M KSO, 5 m M NaN, 5 m M MgCl, 5 m M potassium oxalate, 0.5 m M EGTA, and different CaClconcentrations to give pCa values between 5.4 or 7.5; free calcium concentrations were calculated by the method of Fabiato and Fabiato (1979). The calcium uptake mixture also contained 1.0 µ M each of carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and valinomyocin to dissipate gradients of pH and membrane potential, respectively (Levy et al., 1992). Calcium uptake was initiated by the addition of 5 m M ATP. Aliquots of 250 µl (method I) or 100 µl (method II) were filtered at various time points on 0.22-µm filters (GS type, Millipore) and washed twice with 3 ml of 10 m M MOPS (pH 7.0), 100 m M KSO, 10 m M MgCl, and 5 m M LaCl. Quantitation of Ca was done by scintillation counting. The initial rates of calcium uptake were calculated by least-square regression of the 0.5-, 1.0-, and 2.0-min values for pCa 5.4 or the 2-, 4-, and 6-min values for pCa 6.8.

Cardiac SR was incubated with PLB antibody at a weight ratio of 2:1 (SR protein/antibody) for 20-30 min on ice. Control samples contained an equivalent volume of antibody buffer. Calcium uptake was measured in the same way as method II with 80 µg of cardiac SR in 0.75 ml of assay solution. In this case, no carbonyl cyanide p-trifluoromethoxy-phenylhydrazone or valinomycin was added.

Phosphate release from ATP was measured by the malachite green procedure (Lanzetta et al., 1979). This assay was conducted concurrently with the calcium uptake assay by adding 100-µl aliquots of uptake mixture to 400 µl of malachite green reagent. The absorbance was read at 660 nm, and Prelease was calculated by comparison of values to standard curves generated with known amounts of P. Because purified Ca-ATPase was used, calcium-independent ATPase activity was negligible and therefore was not taken into consideration.

The permeability of vesicles to calcium was determined by monitoring the calcium-sensitive fluorescence of Fluo 3 trapped inside vesicles during reconstitution. Proteoliposomes were reconstituted at a lipid concentration of 10 mg/ml in a buffer containing 50 m M KSO, 10 m M imidazole (pH 7.0), 200 µ M Fluo 3 (Molecular Probes), and 30 µ M CaCl; the lipid-to-Ca-ATPase weight ratio was 80:1 and, when required, PLB or PLBwas included at a 8:1 molar ratio with Ca-ATPase. For fluorescence measurements, 50 µl of proteoliposomes were diluted into 2 ml of a solution containing 50 m M KSOand 10 m M imidazole (pH 7.0). The excitation wavelength was 488 nm and the emission wavelength was 537 nm; a small slit was used to prevent bleaching of Fluo 3, which could be observed at high excitation intensities. Fluorescence from four samples was monitored simultaneously at 1-min intervals at 25 °C with stirring. Stoichiometry of PLB and Ca-ATPase in Vesicles Reconstituted vesicles were separated from unincorporated PLB and Ca-ATPase using sucrose density gradient centrifugation. After reconstitution, 100 µl of vesicles were mixed with 100 µl of a solution containing 60% sucrose, 100 m M KSO, 10 m M PIPES (pH 7.0), and 0.5 mg/ml Triton X-100; this small amount of detergent was necessary to allow equilibration of sucrose within the vesicles (Levy et al., 1992), but did not solubilize the vesicles. This mixture was loaded at the bottom of a 0.8-ml centrifuge tube and overlaid with a stepwise gradient containing 20, 15, 10, 5, and 2.5% sucrose dissolved in 10 m M PIPES (pH 7.0) and 100 m M KSO(0.1 ml for each layer). The gradient was centrifuged for 16 h at 135,000 g (Beckman, SW55 Ti rotor). Fractions of 100 µl were collected from the bottom of the tube and subjected to immunoblot analysis. SDS-PAGE was performed with 6-20% gradient polyacrylamide gels, and proteins were transferred to polyvinylidene difluoride membranes (Bio-Rad) in 25 m M Tris, 193 m M glycine, pH 8.3, 10% methanol, and 0.01% SDS at 4 °C for 1 h at 200 mA constant current. After transfer and blocking, the membranes were incubated with monoclonal antibodies either to PLB (2D12) or to Ca-ATPase (V6E8 from Dr. Kevin Campbell) in blocking buffer. The membrane was washed and incubated for 1 h with goat, anti-mouse antibody conjugated to horseradish peroxidase (Fisher Scientific). After washing with binding buffer, the immunoreactive protein bands were developed with 3,3`-diaminobenzidine tetrahydrochloride reagent (Sigma). Immunoblots were digitized with a gel densitometer (Bio-Rad) in the reflectance mode, and bands were quantitated with the associated image analysis software. For Ca-ATPase, a box was drawn around individual bands, whereas for PLB, the entire lane was selected to include all oligomeric forms of the molecule. After subtracting background, the density was integrated within each box; this integrated density is linearly related to the amount of protein present in each band.


RESULTS

Milligram quantities of highly purified, recombinant PLB were easily obtained in one purification step from carbonate-extracted insect cells using monoclonal antibody affinity chromatography. Fig. 1 documents the purity of the recombinant PLB by SDS-PAGE. A major band of approximately 25 kDa (PLB pentamer) was observed. As is characteristic for native PLB, tetrameric through monomeric PLB was also observed, especially after boiling the sample in SDS prior to electrophoresis. The mobility of recombinant PLB on SDS-PAGE was identical to that obtained with canine cardiac PLB (Fig. 1). In other experiments, we determined that recombinant PLB had a blocked N terminus, as is found for canine cardiac PLB. The amino acid sequence of recombinant PLB was confirmed after cleaving the protein at lysine residue 3 with endoproteinase Lys-C followed by automated sequence analysis (Palmer et al., 1991). Sequencing through 26 consecutive PLB residues revealed no extraneous protein sequence. Recombinant PLB also exhibited the characteristic mobility shift on SDS-PAGE (Wegener and Jones, 1984) induced after phosphorylation with the catalytic subunit of cAMP-dependent protein kinase (data not shown).

Using this purified, recombinant PLB, we developed methods for reconstitution with purified Ca-ATPase from skeletal muscle. Our assay for regulation was to compare calcium uptake in the presence and absence of a monoclonal antibody to PLB, which was previously shown to produce the same effect as PLB phosphorylation; that is, to relieve the inhibition of Ca-ATPase (Briggs et al., 1992; Cantilina et al., 1993; Sham et al., 1991, see also Kimura et al., 1991; Morris et al., 1991). In cardiac SR, the PLB antibody shifts the calcium-dependent stimulation of Ca-ATPase toward lower calcium concentrations (Fig. 2, left panel), suggesting an increase in apparent calcium affinity by Ca-ATPase. At pCa 6.8, the stimulation is large, 15-fold for cardiac SR, whereas at pCa 5.4 there is no stimulation at all. For our reconstituted preparations, we obtained a similar shift (Fig. 2, right panel), and we routinely sampled this curve at two pCa values: 6.8 and 5.4 ( arrows in Fig. 2).


Figure 2: Calcium-dependence of calcium uptake for cardiac SR vesicles ( left panel) and for vesicles reconstituted with skeletal muscle Ca-ATPase plus PLB ( right panel). PLB was reconstituted at a 8:1 molar ratio according to method II. Calcium uptake was measured in the presence () and absence () of monoclonal antibody (2D12) to PLB. The arrows indicate the free calcium concentrations selected to characterize the various reconstitutions in Fig. 3 and Table II.



We characterized two methods for coreconstitution of PLB with Ca-ATPase, which differ in the preparation of lipid and the way PLB is added to the lipid. We found that when PLB and Ca-ATPase were added to detergent-solubilized lipid vesicles at the same time (method I under ``Materials and Methods:''), PLB inhibited calcium uptake by only 20% relative to control levels at low ionized calcium concentration (pCa 6.8, Table II). However, when PLB was first mixed with lipid in a hydrophobic environment (method II under ``Materials and Methods''), and detergent-solubilized Ca-ATPase was later added to the resulting proteoliposomes, PLB inhibited calcium uptake by 50% ( and Fig. 3). In both cases, the inhibition by PLB was fully reversed by adding PLB monoclonal antibody. All subsequent reconstitutions were done with the second, more successful method. As shown in Fig. 4, the maximal effect was obtained when PLB was reconstituted with Ca-ATPase at a molar ratio of 8:1. The effect did not occur at pCa 5.4, which provides the maximal rate of calcium uptake and therefore represents Vfor Ca-ATPase. These effects on calcium uptake were mirrored by those on ATPase activity ( and Fig. 3 ) suggesting that PLB affected the turnover rate of Ca-ATPase rather than the permeability of the vesicles to calcium.


Figure 3: Calcium uptake rates and ATPase activities measured from proteoliposomes of Ca-ATPase coreconstituted with PLB, PLB, or incubated with PLB. The data correspond to those in Table II except that uptake rates and ATPase activities were calculated as the percentage of control ( ctrl), defined as proteoliposomes assayed with no added PLB or PLB antibody. The molar ratio of PLB/Ca-ATPase and PLB/Ca-ATPase was 8:1, whereas PLB/Ca-ATPase was 100:1 ( i.e. 100 µ M during incubation and subsequently diluted to 7 µ M for the assays). These molar ratios were based on a PLB monomer of M= 6080 and a Ca-ATPase monomer of M= 115,000. Reconstitutions with intact PLB were done by method II. Error bars represent the S.E. from three to nine experiments. In some cases, the error is too small for the bar to be visible; in the case of the control, however, because it is defined as 100% there is no associated error.



We used this reconstitution method to compare the effects of purified, canine cardiac PLB (Jones et al., 1985) on skeletal muscle Ca-ATPase with those of recombinant PLB. We found that the effects of the two kinds of PLB were indistinguishable. In particular, proteoliposomes with canine PLB gave uptake rates at pCa 6.8 of 0.19 µmol/mg/min compared with 0.37 µmol/mg/min for controls lacking PLB; incubation with antibody produced uptakes of 0.37 and 0.35 µmol/mg/min for proteoliposomes with and without PLB, respectively. This 2-fold stimulation is comparable to results with recombinant PLB (shown in ). Uptake rates at pCa 5.8 with canine PLB (4.4-4.7 µmol/mg/min) were also comparable to those obtained with recombinant PLB and, as expected, there was no significant effect of the antibody on the uptake rates at this pCa.

We also tested whether PLB altered the calcium permeability of vesicles by monitoring leakage of calcium out of vesicles reconstituted with various combinations of PLB, PLB, and Ca-ATPase. To do this, we measured the calcium-dependent fluorescence of Fluo 3, which had been trapped inside the vesicles during reconstitution (Fig. 5). Differences were observed in the initial levels of fluorescence, which most likely reflect different amounts of trapped Fluo 3 and not differences in internal calcium concentrations. Two factors contribute to these differences: 1) because Fluo 3 was adsorbed by the Biobeads during reconstitution, differences in the rates of vesicle closure will affect the internal concentration of Fluo 3, and 2) vesicle size determines total internal volume and this size may vary for a variety of ill-defined reasons. We compensated for these differences by replotting data as the percentage of initial fluorescence, in order to compare the rates of leakage from different populations of vesicles. Thus, Fig. 5shows that pure lipid vesicles are least permeable, followed closely by vesicles containing Ca-ATPase together with either PLB or PLB. Vesicles containing only Ca-ATPase or PLB or PLBwere the most permeable.


Figure 5: Permeability of reconstituted proteoliposomes to calcium. A typical experiment is illustrated by the fluorescence signals in the inset. At time 0, vesicles were diluted 20-fold into a calcium-free buffer. The initial fluorescence reflects Fluo 3 and calcium trapped inside vesicles (Ca) as well as a smaller signal from diluted calcium (Ca) and Fluo 3 outside vesicles. EGTA was then added to make the concentration of free Caextremely low (pCa > 8); this served to eliminate any contribution from Fluo 3 outside the vesicles and maintained a maximal calcium gradient across the vesicular membrane (note that the brief drop in fluorescence to near zero is an artifact of the shutter closing when the lid was opened for addition of EGTA). The subsequent, steady decline in fluorescence reflects the decrease in Ca, or the leakage of calcium across the reconstituted bilayer. After 1 h, 4 µg of a calcium ionophore, A23187, was added to verify that this signal truly reflected Caand to give a base line for normalization; in all cases, the subsequent fluorescent signal was close to the dark signal (which is shown as the near-zero signal when the lid is lifted to add the A23187). For comparison of different preparations, the base line was subtracted and data were replotted as the percent of fluorescence immediately after EGTA addition, which reflects the initial concentration of Ca. The normalized data shown represents an average of two different experiments, and the rate of decrease corresponds to the permeability of the vesicles.



In light of previous reports, we tested whether either of the two major domains of PLB was sufficient for inhibition of Ca-ATPase in our reconstituted system. The membrane-spanning region, PLB, was generated by trypsinolysis of the purified protein and has been previously shown to stabilize the pentamer (Fujii et al., 1989; Simmerman et al., 1986). We found that this peptide inhibited calcium pumping at both low and high calcium concentrations, but that ATPase activities were unaffected ( and Fig. 3). Since PLBdoes not increase the calcium permeability of vesicles reconstituted with Ca-ATPase (Fig. 5), the results suggest that PLBpartially uncouples ATP hydrolysis from calcium transport. We also added the synthetic, cytoplasmic domain, PLB, to reconstituted preparations of Ca-ATPase. However, we observed no effect of PLB, at a molar excess of 100:1 relative to Ca-ATPase, on either calcium uptake or ATPase activity regardless of calcium concentration ( and Fig. 3 ). At even higher molar ratios (up to 300:1), the peptide actually stimulated calcium uptake slightly at pCa 6.8, but had no effect at pCa 5.4 (data not shown).

To address the stoichiometry of the interaction between PLB and Ca-ATPase, we quantitated the efficiency of incorporation of the two proteins into lipid vesicles. First, membrane vesicles were separated from unincorporated protein by sucrose density gradient centrifugation, and protein was tracked by immunoblotting (Fig. 6). We found that vesicles equilibrated at the top of the gradient and contained virtually all of the Ca-ATPase. In contrast, PLB was dispersed more broadly throughout the gradient. In particular, most of the PLB was detected at the top and bottom of the gradient; i.e. both with the vesicular material and as a ``pellet'' of dense material in 30% sucrose. Thus, the 8:1 molar ratio of PLB:Ca-ATPase used for reconstitution is actually 3:1 within the recovered membranes.


DISCUSSION

Reconstituted Systems versus Native SR

PLB is widely regarded as the major regulatory component of Ca-ATPase in cardiac SR, yet no one has been entirely successful in recreating, in reconstituted systems, the large functional effect that this protein has on Ca-ATPase in intact SR. One very good reason has been the difficulty in obtaining sufficient quantities of pure PLB for a thorough investigation of different reconstitution conditions. Our success at expressing and purifying milligram quantities of PLB from Sf21 cells led us not only to investigate the ability of PLB to regulate Ca-ATPase, but also to try to improve previous methods of coreconstitution.

Indeed, we have demonstrated that purified, recombinant PLB is capable of regulating the skeletal muscle Ca-ATPase and that the magnitude of this regulation is at least comparable to results obtained with other systems. In particular, recombinant PLB inhibited Ca-ATPase to 50% of controls at pCa 6.8, which is consistent with effects obtained by coexpression of PLB with Ca-ATPase in COS-1 cells (Fujii et al., 1990; Toyofuku et al., 1993; Verboomen et al., 1992), by induction of PLB expression in fast-twitch muscle (Briggs et al., 1992) and by our own reconstitutions with native, canine PLB. Our effects are 2.5-fold larger than those previously obtained by reconstitution of native, canine PLB with Ca-ATPase either from skeletal (Szymanska et al., 1990), or cardiac (Kim et al., 1990) muscle. Similar to a recent report on reconstitution of synthetic PLB (Vorherr et al., 1993), we found that the regulatory effects depend on the method used to incorporate PLB into the membrane. Our first method, involving full solubilization of PLB, Ca-ATPase, and lipid followed by detergent removal with Biobeads, produced only minimal effects on calcium uptake. This method was originally presented and thoroughly characterized by Rigaud and co-workers (Levy et al., 1990a, 1990b, 1992) and gives excellent rates of calcium uptake (see and Fig. 2, right panel). However, independent measurements on our vesicles by infrared spectroscopy indicated that PLB incorporation into the membranes was very inefficient by this method (Tatulian et al., 1995). A more successful method involved mixing PLB with lipid in a non-aqueous environment followed by formation of PLB-containing vesicles; Ca-ATPase was later added to these vesicles in detergent and the detergent was removed by Biobeads. Even employing this improved method, we found that only 40% of PLB was associated with lipid vesicles after reconstitution. Reasons for the failure of PLB to insert into the membranes could be due either to improper folding of the peptide or to nonspecific protein aggregation, perhaps as the detergent concentration falls during treatment with Biobeads. In any case, this difficulty in incorporating PLB into membranes may partially explain why such high stoichiometries of PLB to Ca-ATPase are often necessary to observe regulatory effects.

Apart from the question of stoichiometry, the maximal effects of PLB in all artificial systems have always been much smaller than those in cardiac SR, where calcium uptake is stimulated many fold, either by phosphorylation of PLB (Kranias, 1985; Tada et al., 1974) or by addition of PLB antibodies (Briggs et al., 1992; Cantilina et al., 1993; Kimura et al., 1991; Morris et al., 1991). This has been true for reconstituted systems (Kim et al., 1990; Sasaki et al., 1992; Szymanska et al., 1990; Vorherr et al., 1993), for genetically engineered cell culture (Fujii et al., 1990; Toyofuku et al., 1993; Verboomen et al., 1992), and for non-cardiac muscles that have been chronically stimulated to induce PLB expression (Briggs et al., 1992). This failure to mimic the magnitude of the effect observed with cardiac SR could be related either to the composition of the bilayer ( e.g. a requirement for a particular lipid composition or an optimal lipid-to-protein ratio) or to additional, unknown factors ( e.g. a requirement for some unknown protein component in cardiac SR or for some special control over assembly of PLB with Ca-ATPase during biogenesis of SR). Given our success in expressing and purifying recombinant PLB and our demonstration that this recombinant PLB functions like native, canine PLB, we can now begin to address some of these issues more directly.

PLB Affects Calcium Affinity of Ca-ATPase

One of our main results is that PLB regulates the apparent calcium affinity of Ca-ATPase, but does not alter the V. This conclusion is clearly indicated by Fig. 2and further supported by the data in . In particular, neither calcium uptake nor ATPase activity was affected by PLB at pCa 5.4, where calcium transport is maximal, but PLB did inhibit both of these activities at pCa 6.8, where calcium transport is suboptimal. This inhibitory effect was reversed by the PLB monoclonal antibody, similar to results obtained with native SR vesicles (Briggs et al., 1992; Jones and Field, 1993) or with isolated cardiac myocytes (Sham et al., 1991). Our results are consistent with those from a recent study comparing SR from cardiac and skeletal muscle (Cantilina et al., 1993) in which PLB was found to affect only the apparent calcium affinity. These authors specifically attributed this effect to a slowing of a crucial conformational change during calcium binding by Ca-ATPase. A possible mechanism for this effect is suggested by recent results with time-resolved phosphorescence anisotropy, suggesting that PLB slows this conformational change by promoting aggregation of molecules of Ca-ATPase (Voss et al., 1994). These conclusions conflict with those from earlier reconstitution studies (Sasaki et al., 1992), which suggested that PLB also inhibits Ca-ATPase at saturating calcium concentrations. However, in this earlier study, very high molar ratios (>100:1) of PLB/Ca-ATPase were required and only ATPase activities were measured; an increase in membrane permeability was therefore not excluded. Finally, reconstitutions by Kranias and co-workers (Kim et al., 1990; Szymanska et al., 1990) were characterized at only a single calcium concentration (pCa 6.0), where it is impossible to distinguish effects on calcium affinity from those on V.

Our results do not support the idea that PLB generates a passive calcium leak across cardiac SR membranes (Kovacs et al., 1988) because changes in calcium uptake correlated with changes in ATPase activity. Furthermore, direct measurements of permeability showed that proteoliposomes containing both Ca-ATPase and PLB were, if anything, less permeable than those containing Ca-ATPase alone.

Neither of the peptides that we studied, representing the two major domains of PLB, were able to mimic the full physiological effect of intact PLB on Ca-ATPase. In particular, the membrane-spanning domain, which is reported to stabilize pentamer formation, appeared to uncouple calcium transport from ATPase activity and the cytoplasmic domain, which contains sites for phosphorylation, had no effect at all. According to our measurements of membrane permeability, this uncoupling effect of PLBis not due to increased calcium leakage. A possible explanation is that PLBinteracts directly with Ca-ATPase and somehow partially uncouples ATPase hydrolysis from calcium transport, although we have no direct evidence to support this mechanism. Previous studies with a similar peptide, PLB, showed an effect on the calcium affinity of Ca-ATPase (Sasaki et al., 1992), but again, very high molar ratios of PLB to Ca-ATPase were required and calcium transport was not measured. In previous investigations of the cytoplasmic domain, investigators using either reconstituted systems (Kim et al., 1990; Sasaki et al., 1992) or skeletal SR (Hughes et al., 1994) have reported that their peptides inhibit Ca-ATPase, whereas other investigators using either cardiac atrial SR (Jones and Field, 1993) or skeletal SR (Vorherr et al., 1992) showed that their peptides had no effect. Considering that comparable concentrations of rather similar peptides were used in all of these studies, it is not clear why results have been so variable. Minor peptide impurities represent one possibility, given the very high concentrations of peptides used in the studies that show an effect; such impurities could potentially act either on Ca-ATPase or on the lipid bilayer. Additionally, the different experimental protocols are not entirely consistent, some measuring ATPase activity and others calcium uptake-sometimes at only a single calcium concentration. For this reason, we suggest that it is important for future studies to fully characterize effects of PLB peptides on Ca-ATPase by measuring calcium uptake and ATPase activity at several different free calcium concentrations.

Physical Basis for Regulation

The use of highly purified preparations of PLB and Ca-ATPase makes it possible to calculate the stoichiometry required for a functional interaction between the two proteins. Under conditions generating the maximal effect, we found a molar stoichiometry of 3:1 in the reconstituted membranes, which is close to the 5:1 ratio expected if each PLB pentamer were bound to only one Ca-ATPase monomer. This represents an upper limit for this stoichiometry, since not every PLB molecule in the vesicles is necessarily interacting with a Ca-ATPase molecule. In fact, results from time-resolved phosphorescence anisotropy have demonstrated PLB-mediated aggregation of Ca-ATPase (Voss et al., 1994), suggesting that the physical basis for regulation may involve a lower stoichiometry. Future studies will concentrate on increasing the protein-to-lipid ratio and in particular on crystallizing the resultant material within the plane of the membrane. In this way, we hope to obtain more direct structural information regarding the important interaction between Ca-ATPase and PLB.

  
Table: Amino acid analysis of PLB


  
Table: Activity of Ca-ATPase after reconstitution with PLB or its fragments

The monoclonal antibody 2D12 (Ab) was included in the assay as indicated. PLB and Control designate vesicles reconstituted with and without PLB, respectively. The molar ratios used were: 8:1 for PLB/Ca-ATPase and PLB/Ca-ATPase; 100:1 for PLB/Ca-ATPase. pCa 6.8 and pCa 5.4 indicate the ionized calcium concentrations used for the activity assays, as defined by the arrows in Fig. 2. Values represent the mean ± S.E. of three to eight experiments.



FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL48807 (to D. L. S.), HL49428 and HL06308 to (L. R. J.), and American Heart Association Fellowship VA-93-F16 (to S. A. T.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed.

The abbreviations used are: PLB, phospholamban; SR, sarcoplasmic reticulum; PLB, peptide composed of amino acid residues 1-31 of PLB; PLB, peptide composed of amino acid residues 26-52 of PLB; CE, octaethylene glycol monododecyl ether; -OG, -octyl glucoside; EYPC, egg yolk phosphatidyl choline; EYPA, egg yolk phosphatidic acid; PAGE, polyacrylamide gel electrophoresis; rpm, revolutions/min; MOPS, 4-morpholinepropanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid.


ACKNOWLEDGEMENTS

We gratefully acknowledge Dr. Kevin Campbell for providing the monoclonal antibody to skeletal Ca-ATPase (V6E8). We also thank Bruce Scott for providing the canine cardiac PLB cDNA clone, Ron Pace for excellent technical assistance, and Carol Fiol for making the synthetic PLB peptide.


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