From the
Phospholamban (PLB) is a small, transmembrane protein that
resides in the cardiac sarcoplasmic reticulum (SR) and regulates the
activity of Ca
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
To study the specific
effects of PLB on the reaction cycle of Ca
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
Preparation of Ca
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
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
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
Proteoliposomes
containing PLB
Because PLB
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.
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 P
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 K
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
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
To address the stoichiometry of
the interaction between PLB and Ca
Indeed, we have demonstrated that
purified, recombinant PLB is capable of regulating the skeletal muscle
Ca
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
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
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
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
We gratefully acknowledge Dr. Kevin Campbell for
providing the monoclonal antibody to skeletal
Ca
ABSTRACT
INTRODUCTION
MATERIALS and METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-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.
-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.
-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.
-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.
-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%
C
E
, 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
10
cells/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 Na
CO
(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.
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).
-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,
C
E
, 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
C
E
and 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).
0.8 ml of CHCl
was 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.
were 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 PLB
in
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.
is 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.
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 K
SO
, 5
m
M NaN
, 5 m
M MgCl
, 5 m
M potassium oxalate, 0.5 m
M EGTA, and different CaCl
concentrations 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 K
SO
, 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.
release
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.
SO
, 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 PLB
was
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 K
SO
and 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 K
SO
, 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 K
SO
(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.
-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 V
for 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.
, 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
PLB
were 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 Ca
extremely 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 Ca
and 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 PLB
does not increase the calcium permeability of vesicles
reconstituted with Ca
-ATPase (Fig. 5), the
results suggest that PLB
partially 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).
-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.
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.
-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.
-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
One of our main results is that PLB
regulates the apparent calcium affinity of Ca-ATPase
-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
.
-ATPase and PLB
were, if anything, less permeable than those containing
Ca
-ATPase alone.
-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 PLB
is not due to increased calcium
leakage. A possible explanation is that PLB
interacts 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
-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.
, peptide composed
of amino acid residues 1-31 of PLB; PLB
,
peptide composed of amino acid residues 26-52 of PLB;
C
E
, 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.
-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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.