Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to β-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (∼1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by ∼50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport. Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to β-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (∼1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by ∼50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport.
Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by β-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitroco-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 °C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 °C, has no inhibitory activity when assayed at 25 °C. However, at 37 °C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 °C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase. Phospholamban (PLB), a 52-amino acid integral membrane protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum through PLB phosphorylation mediated by β-adrenergic stimulation. Based on site-directed mutagenesis and coexpression with Ca-ATPase (SERCA2a) in Sf21 insect cells or in HEK 293 cells, and on spin label detection of PLB oligomeric state in lipid bilayers, it has been proposed that the monomeric form of PLB is the inhibitory species, and depolymerization of PLB is essential for its regulatory function. Here we have studied the relationship between PLB oligomeric state and function by in vitroco-reconstitution of PLB and its mutants with purified Ca-ATPase. We compared wild type-PLB (wt-PLB), which is primarily a pentamer on SDS-polyacrylamide gel electrophoresis (PAGE) at 25 °C, with two of its mutants, C41L-PLB and L37A-PLB, that are primarily tetramer and monomer, respectively. We found that the monomeric mutant L37A-PLB is a more potent inhibitor than wt-PLB, supporting the previous proposal that PLB monomer is the inhibitory species. On the other hand, C41L-PLB, which has a monomeric fraction comparable to that of wt-PLB on SDS-PAGE at 25 °C, has no inhibitory activity when assayed at 25 °C. However, at 37 °C, a 3-fold increase in the monomeric fraction of C41L-PLB on SDS-PAGE resulted in inhibitory activity comparable to that of wt-PLB. Upon increasing the temperature from 25 to 37 °C, no change in fraction monomer or inhibitory activity for wt-PLB and L37A-PLB was observed. Based on these results, the extent of inhibition of Ca-ATPase by PLB or its mutants appears to depend not only on the propensity of PLB to dissociate into monomers but also on the relative potency of the particular PLB monomer when interacting with the Ca-ATPase. phospholamban wild type native phospholamban expressed in Sf21 insect cell sarcoplasmic reticulum a monoclonal antibody to PLB (2D12) polyacrylamide gel electrophoresis β-octyl glucoside egg yolk phosphatidylcholine egg yolk phosphatidic acid dioleoyl phosphatidylcholine dioleoyl phosphatidylethanolamine 3,3′-diaminobenzidine tetrahydrochloride polyvinylidene difluoride N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine 4-morpholinepropanesulfonic acid sarco (endo) plasmic reticulum Ca-ATPase Phospholamban (PLB)1 is a small membrane protein of 52 amino acid residues, which is composed of an N-terminal cytoplasmic domain and a C-terminal membrane-spanning domain (1Simmerman H.K.B. Collins J.H. Theibert J.L. Wegener A.D. Jones L.R. J. Biol. Chem. 1986; 261: 13333-13341Abstract Full Text PDF PubMed Google Scholar, 2Fujii J. Ueno A. Kitano K. Tanaka S. Kadoma M. Tada M. J. Clin. Invest. 1987; 79: 301-304Crossref PubMed Scopus (137) Google Scholar). In response to β-adrenergic stimulation, PLB phosphorylation regulates the rate of calcium pumping by the Ca-ATPase of cardiac sarcoplasmic reticulum (3Lindemann J.P. Jones L.R. Hathaway D.R. Henry B.G. Watanabe A.M. J. Biol. Chem. 1983; 258: 464-471Abstract Full Text PDF PubMed Google Scholar, 4Colyer J. Cardiovasc. Res. 1993; 27: 1766-1771Crossref PubMed Scopus (52) Google Scholar, 5Stokes D.L. Cur. Opin. Struct. Biol. 1997; 7: 550-556Crossref PubMed Scopus (33) Google Scholar, 6Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar). Although the expression of PLB is largely restricted to cardiac SR, to a lesser extent its presence is reported in slow-twitch skeletal muscle SR (7Briggs F.N. Lee K.F. Wechsler A.W. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar, 8Kirchberger M.A. Tada M. J. Biol. Chem. 1976; 251: 725-729Abstract Full Text PDF PubMed Google Scholar) and smooth muscle SR (9Raeymaekers L. Eggermont J.A. Wuytack F. Casteels R. Cell Calcium. 1990; 11: 261-268Crossref PubMed Scopus (68) Google Scholar). In its unphosphorylated form, PLB inhibits the rate of Ca2+ pumping and ATP hydrolysis by the Ca-ATPase at subsaturating [Ca2+]. Upon phosphorylation of PLB by cAMP-dependent protein kinase A or Ca2+/calmodulin-dependent protein kinase II, this inhibition is relieved (4Colyer J. Cardiovasc. Res. 1993; 27: 1766-1771Crossref PubMed Scopus (52) Google Scholar, 6Simmerman H.K.B. Jones L.R. Physiol. Rev. 1998; 78: 921-947Crossref PubMed Scopus (467) Google Scholar, 10Tada M. Kadoma M. BioEssays. 1989; 10: 163-169Crossref PubMed Scopus (60) Google Scholar). Although PLB is not present in fast-twitch muscle (8Kirchberger M.A. Tada M. J. Biol. Chem. 1976; 251: 725-729Abstract Full Text PDF PubMed Google Scholar, 11Jones L.R. Simmerman H.K.B. Wilson W.W. Gurd F.R.N. Wegener A.D. J. Biol. Chem. 1985; 260: 7721-7730Abstract Full Text PDF PubMed Google Scholar, 12Jorgensen A.O. Jones L.R. J. Cell Biol. 1986; 104: 1343-1352Crossref Scopus (32) Google Scholar), it has been shown to be capable of regulating the fast-twitch Ca-ATPase isoform (SERCA1) in both reconstitution (13Kim H.W. Steenaart N.A.E. Ferguson D.G. Kranias E.G. J. Biol. Chem. 1990; 265: 1702-1709Abstract Full Text PDF PubMed Google Scholar, 14Szymanska G. Kim H.W. Cuppoletti J. Kranias E.G. Membr. Biochem. 1991; 9: 191-202Crossref Scopus (20) Google Scholar, 15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar) and coexpression (16Toyofuku T. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1993; 268: 2809-2815Abstract Full Text PDF PubMed Google Scholar) experiments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and low angle laser light scattering in SDS solutions (17Wegener A.D. Jones L.R. J. Biol. Chem. 1984; 259: 1834-1841Abstract Full Text PDF PubMed Google Scholar, 18Watanabe Y. Kijima Y. Kadoma M. Tada M. Takagi T. J. Biochem. (Tokyo). 1991; 110: 40-45Crossref PubMed Scopus (23) Google Scholar) showed that PLB is predominantly a homopentamer in equilibrium with a small fraction as monomer. Systematic replacement of single hydrophobic amino acid residues, Leu or Ile, by Ala in the membrane-spanning region transformed PLB from pentameric to monomeric form on SDS-PAGE (19Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). Based on these results, a model for a tightly packed coiled-coil pentamer (19Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar, 20Arkin I.T. Adams P.D. MacKenzie K.R. Lemmon M.A. Brunger A.T. Engelman D.M. EMBO J. 1994; 13: 4757-4764Crossref PubMed Scopus (174) Google Scholar, 21Karim C.B. Stamm J.D. Karim J. Jones L.R. Thomas D.D. Biochemistry. 1998; 37: 12074-12081Crossref PubMed Scopus (53) Google Scholar) has been proposed: the α-helical transmembrane domains of five monomers associate by intramembrane Leu/Ile interactions forming a Leu/Ile zipper. Monomeric mutants of PLB were shown to be more potent inhibitors of the cardiac isoform of the Ca-ATPase (SERCA2a) when coexpressed in a heterologous system (22Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Kimura Y. Kurzydlowski M. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Crossref PubMed Scopus (249) Google Scholar, 24Autry J.M. Jones L.R. Ann. N. Y. Acad. Sci. 1998; 853: 92-102Crossref PubMed Scopus (14) Google Scholar). By using EPR spectroscopy, it was shown that PLB exists in an average oligomeric size of 3.5 in DOPC vesicles, and upon phosphorylation the average oligomeric size increased to 5.3 (26Cornea R.L. Jones L.R. Autry J.M. Thomas D.D. Biochemistry. 1997; 36: 2960-2967Crossref PubMed Scopus (160) Google Scholar). This study suggested the existence of a dynamic equilibrium between PLB subunits in the lipid bilayer and that the regulation of the oligomeric state of PLB is critical for its regulation of the Ca-ATPase. This led to the hypothesis that the active inhibitory species of PLB is the monomer and that increased oligomerization of PLB upon phosphorylation contributes to relief of the inhibition (22Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Kimura Y. Kurzydlowski M. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Crossref PubMed Scopus (249) Google Scholar, 25Thomas D.D. Reddy L.G. Karim C.B. Li M. Cornea R. Autry J.M. Jones L.R. Stamm J. Ann. N. Y. Acad. Sci. 1998; 853: 186-195Crossref PubMed Scopus (40) Google Scholar, 26Cornea R.L. Jones L.R. Autry J.M. Thomas D.D. Biochemistry. 1997; 36: 2960-2967Crossref PubMed Scopus (160) Google Scholar). However, there are several unanswered questions as follows. (a) So far only pentamer/monomer (e.g. native PLB) and purely monomeric (e.g. L37A-PLB) structural mutants of PLB and their functional effects have been reported (22Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Kimura Y. Kurzydlowski M. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Crossref PubMed Scopus (249) Google Scholar), but this does not exclude the possibility of other oligomeric forms of PLB as inhibitory species. (b) In a coexpression system the relative ratios of PLB and Ca-ATPase may vary, so it is possible that the apparent enhanced inhibition of the pump by the monomeric mutants is due to variation in expression levels of PLB mutants and Ca-ATPase. In the present study, we have tested the inhibitory function of recombinant wild type-PLB (wt-PLB) and two different structural mutants, a phospholamban monomer and tetramer (19Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar), using an in vitro co-reconstitution system (15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Reddy L.G. Jones L.R. Pace R.C. Stokes D.L. J. Biol. Chem. 1996; 271: 14964-14970Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). We have characterized the inhibitory function of these structural mutants as a function of increasing molar ratios of PLB/Ca-ATPase and as a function of temperature. The correlation of these results with the oligomeric states of these mutants, as indicated by SDS-PAGE, provides new insight into the structural basis of Ca-ATPase regulation by PLB. The reagents used were octaethylene glycol monododecyl ether (C12E8), β-octyl glucoside (β-OG), and Biobeads SM2 were purchased from Calbiochem (San Diego, CA). Dioleoyl phosphatidylcholine (DOPC), dioleoyl phosphatidylethanolamine (DOPE), egg yolk phosphatidylcholine (EYPC), and egg yolk phosphatidic acid (EYPA) were obtained from Avanti Polar Lipids (Alabaster, AL). The reagents for SDS-PAGE (16.5% Tris/Tricine ready gels and the Tris/Tricine gel running buffer) and the PVDF membrane used for immunoblots were from Bio-Rad. Anti-PLB monoclonal antibody (2D12) was prepared as described previously (7Briggs F.N. Lee K.F. Wechsler A.W. Jones L.R. J. Biol. Chem. 1992; 267: 26056-26061Abstract Full Text PDF PubMed Google Scholar, 15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 28Sham J.S.K. Jones L.R. Morad M. Am. J. Physiol. 1991; 261: H1344-H1349PubMed Google Scholar). Horseradish peroxidase-coupled goat anti-mouse antibody was supplied by Fisher (Southern Biotechnology Laboratories, Inc.). The substrate for horseradish peroxidase, 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent, and Reactive Red affinity column were purchased from Sigma. All the other reagents were of highest purity available and were purchased from Sigma. Recombinant wt-PLB and the mutants were expressed in Sf21/baculovirus insect cell system and purified by monoclonal antibody (2D12) affinity column chromatography as described previously (15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 19Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The concentration of PLB was determined by the Amido Black assay (29Schaffner W. Weissman C. Anal. Biochem. 1973; 56: 502-514Crossref PubMed Scopus (1954) Google Scholar). The purified protein was stored at −70 °C at a protein concentration of 1.5–2.5 mg/ml, in a buffer (“OG buffer”) containing 88 mm MOPS, 18 mm glycine, 5 mm dithiothreitol, and 0.92% β-octyl glucoside (β-OG) at pH 7.2. Rabbit skeletal sarcoplasmic reticulum (SR) Ca-ATPase was purified from light SR using the reactive-red affinity chromatography method (30Stokes D.L. Green N.M Biophys. J. 1990; 57: 1-14Abstract Full Text PDF PubMed Scopus (117) Google Scholar). The method used for functional reconstitution of Ca-ATPase with PLB has been described previously (15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Reddy L.G. Jones L.R. Pace R.C. Stokes D.L. J. Biol. Chem. 1996; 271: 14964-14970Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In short, the required amount of PLB (5–25 μg) in OG buffer was dried (lyophilized) and solubilized in 80 μl of CHCl3 containing 20 μl of 2,2,2-trifluoroethanol and 0.8 mg of DOPC/DOPE (20% DOPE, by weight) for ATP hydrolysis measurements, or EYPC/EYPA (20% EYPA, by weight) for Ca2+ uptake measurements. The solvent was dried under nitrogen and the residual solvent was removed by pumping the sample under vacuum. The dried film of lipid and PLB was hydrated with 50 μl of 20 mmimidazole, pH 7.0, by vortexing throughly followed by a brief sonication. The resulting vesicles, containing lipid and PLB, were made to 20 mm imidazole, pH 7.0, 0.1 m KCl, 5 mm MgCl2, 10% glycerol; then 1.6 mg of β-OG was added followed by 20 μg of purified Ca-ATPase, in a final volume of 100 μl with buffer. For Ca2+ uptake rate measurements, the buffer contained 0.1 m K2 oxalate instead of KCl. The detergent was then removed by incubation with 40 mg of wet Biobeads (25 mg of Biobeads SM2/1 mg of detergent) for 3 h at room temperature. The resultant Ca-ATPase/PLB lipid vesicles were separated from Biobeads and immediately assayed for Ca2+ uptake or ATP hydrolysis activity. Prior to the assay, the reconstituted vesicles were incubated with or without PLB monoclonal antibody, 2D12 (22Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), at a molar ratio of PLB/Ab = 1, for 15 min on ice. 45Ca2+uptake measurements were carried using the microfiltration method (31Martonosi A. Feretos R. J. Biol. Chem. 1964; 239: 648-658Abstract Full Text PDF PubMed Google Scholar) as described previously (15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 27Reddy L.G. Jones L.R. Pace R.C. Stokes D.L. J. Biol. Chem. 1996; 271: 14964-14970Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar) at 25 °C. Each assay was done in duplicate at pCa 5.4 and 6.8 in a volume of 0.25 ml which contained 1–2 μg of Ca-ATPase (10–20 μl of vesicles after the addition of PLB antibody), 50 mm imidazole, pH 7.0, 0.095m K2SO4, 5 mmMgCl2, 5 mm NaN3, 5 mmK2 oxalate, 0.5 mm EGTA, and different concentrations of CaCl2 to obtain pCa values of 5.4 and 6.8; free calcium concentrations were calculated by the method of Fabiato and Fabiato (32Fabiato A. Fabiato F. J. Physiol. (Paris). 1979; 75: 463-505PubMed Google Scholar). The final 45Ca2+in the assay mix was 30 to 50 μCi. The assay mixture also contained 1 μm each of carbonyl cyanidep-trifluoromethoxyphenylhydrazone and valinomycin to dissipate gradients of pH and K+, respectively, and thus to collapse any membrane potential that develops (33Levy D. Gulik A. Bluzart A. Rigaud J.-L. Biochim. Biophys. Acta. 1992; 1107: 283-298Crossref PubMed Scopus (108) Google Scholar). Ca2+uptake was initiated by the addition of 5 mm ATP. Aliquots of 100 μl were filtered at 30 s and 2 min (pCa 5.4) or 6 min (pCa 6.8) after the addition of ATP (we found that these time points gave linear calcium uptake) using 0.22-μm filters (GS type, Millipore) and were washed twice with 20 mm MOPS, pH 7.0, 100 mm K2SO4, 10 mm MgCl2, and 3 mmLaCl3. Quantitation of 45Ca2+ was done by scintillation counting, and the initial calcium uptake rate was calculated from the slope of the line joining the two time points. ATPase activity was assayed by measuring the inorganic phosphate released from the hydrolysis of ATP by Ca-ATPase at 25 and 37 °C, using the method of Lanzetta et al. (34Lanzetta P.A. Alvarez L.J. Reinach P.S. Candia O.A. Anal. Biochem. 1979; 100: 95-97Crossref PubMed Scopus (1821) Google Scholar). Each assay was done in triplicate at pCa 5.4 and 6.8 in a volume of 0.12 ml that contained 0.5–1.0 μg of Ca-ATPase (5–10 μl of vesicles), 50 mm imidazole, pH 7.0, 0.1 m KCl, 5 mm MgCl2, 0.5 mm EGTA, 1–2 μg/ml calcium ionophore (A23187), and different concentrations of CaCl2 to obtain pCa values of 5.4 and 6.8. ATP hydrolysis was initiated by the addition of 2.5 mm ATP. Aliquots of 50 μl were taken at zero time (immediately after the addition of ATP) and at 2 (pCa 5.4) or 6 min (pCa 6.8) and dispensed into 400 μl of malachite green reagent followed by the addition of 50 μl of 34% sodium citrate after 30 s. The absorbance was read at 650 nm, and the rate of ATP hydrolysis was calculated using a standard curve from known concentrations of inorganic phosphate. Reconstituted vesicles were subjected to sucrose gradient centrifugation to separate unincorporated PLB from vesicles containing PLB and Ca-ATPase. After functional assays, 20 μl of the remaining vesicles were loaded on top of a stepwise gradient of 50 (50 μl), 20 (170 μl), 15 (170 μl), 10 (170 μl), and 5% (50 μl) sucrose in 0.8-ml centrifuge tubes. The gradients were centrifuged for 14–16 h at 140,000 ×g (Beckman, SW55 Ti rotor). Fractions of 100–120 μl were collected from the bottom of the tube and subjected to immunoblot analysis. A small volume of each fraction was diluted into transfer buffer (25 mm Tris base, 193 mm glycine, 0.1% SDS, and 10% methanol) and applied to PVDF (polyvinylidene difluoride) membranes (Bio-Rad) using a Slot-Blot apparatus (Bio-Rad). After blocking the membrane with 1% non-fat dry milk, it was incubated for 1 h with monoclonal anti-PLB antibody (2D12). The membrane was washed and incubated for 1 h with goat anti-mouse antibody conjugated to horseradish peroxidase. The membrane was then washed and the immunoreactive protein bands were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Sigma). The immunoblots were scanned by a densitometer using the reflectance mode, and the bands were quantitated using the volume (area × density) analysis method. The densities of the bands were within the linear range of intensities. PLB (wt-PLB) and its mutants, C41L-PLB and L37A-PLB (50–100 ng), were subjected to SDS-PAGE (16.5% Tris/Tricine gels, Bio-Rad) at various temperatures, and proteins were transferred to PVDF membranes in 25 mm Tris (base), 193 mm glycine, pH 8.3, 10% methanol, and 0.1% SDS for 1 h at 4 °C and 200 mA constant current. After blocking the membrane with 1% non-fat dry milk, it was incubated for 1 h with monoclonal anti-PLB antibody (2D12). The membrane was washed and incubated for 1 h with goat anti-mouse antibody conjugated to horseradish peroxidase. The membrane was then washed, and the immunoreactive protein bands were developed with 3,3′-diaminobenzidine tetrahydrochloride (DAB) reagent (Sigma). The immunoblots were scanned by a densitometer using the reflectance mode, and the bands were quantitated using the volume (area × density) analysis method. On SDS-PAGE, wild type PLB (wt-PLB) migrates primarily as a pentamer, with a small fraction as monomer (17Wegener A.D. Jones L.R. J. Biol. Chem. 1984; 259: 1834-1841Abstract Full Text PDF PubMed Google Scholar). The mutants of PLB used in this study, C41L-PLB (Cys 41 to Leu) and L37A-PLB (Leu 37 to Ala), migrated predominantly as tetramer and monomer, respectively, on SDS-PAGE at 25 °C (Fig.1) (19Simmerman H.K.B. Kobayashi Y.M. Autry J.M. Jones L.R. J. Biol. Chem. 1996; 271: 5941-5946Abstract Full Text Full Text PDF PubMed Scopus (223) Google Scholar). The inhibitory function of recombinant PLB (wt-PLB) and its mutants, C41L-PLB and L37A-PLB, was tested in a reconstituted system. Co-reconstitution was done using purified skeletal SR Ca-ATPase and purified PLB in EYPC/EYPA lipid vesicles for Ca2+uptake measurements, and in DOPC/DOPE lipid vesicles for ATP hydrolysis measurements. When EYPC/EYPA or pure DOPC was used for reconstitution, the ATP hydrolysis activity of the enzyme was too low at pCa 6.8 (low calcium) to obtain acceptable precision. Addition of 20% DOPE (by weight) to DOPC enhanced the activity by 40%, providing adequate precision at low calcium. Inhibitory activity of PLB and the mutants, C41L-PLB and L37A-PLB, was assayed by measuring Ca2+ uptake and ATP hydrolysis rates in the presence and absence of anti-PLB monoclonal antibody (2D12). In coexpression studies, the extent of inhibition by PLB or its mutants has usually been reported as a shift (increase or decrease) in K Ca (Ca2+ concentration at which Ca2+ stimulation of activity is half-maximal) compared with the control (no PLB), obtained from a curve of Ca-ATPase activity versus pCa (22Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Kimura Y. Kurzydlowski M. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Crossref PubMed Scopus (249) Google Scholar, 35Kimura Y. Asahi M. Kurzydlowski K. Tada M. MacLennan D.H. J. Biol. Chem. 1998; 273: 14238-14241Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar). In the present study, only two calcium concentrations were used, pCa 5.4 (“high calcium”), where Ca2+ uptake and Ca-ATPase activities are maximal (V max), andpCa 6.8 (“low calcium”), where the shift inK Ca caused by PLB results in a substantial inhibition. Thus at high calcium, only V maxeffects are observed, and at low calcium both effects are observed. We report the extent of inhibition as the percent decrease (percent inhibition) in the Ca-ATPase activity, compared with the activity in the presence of anti-PLB antibody (+PLB-Ab), which is functionally equivalent to PLB phosphorylation and therefore reports the disinhibited SERCA state (15Reddy L.G. Jones L.R. Cala S.E. O'Brian J.J. Tatulian S.A. Stokes D.L. J. Biol. Chem. 1995; 270: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). At 25 °C and pCa 5.4, there is no significant difference in calcium uptake rates in the absence and presence of anti-PLB antibody (Fig. 2, left panel), indicating that PLB or its mutants have no significant effect on the maximal velocity (V max) of the Ca-ATPase. On the other hand, at pCa 6.8 (Fig. 2, right panel), wt-PLB, C41L-PLB, and L37A-PLB inhibit the pump by 48% (0.41 ± 0.10 IU, −PLB-Ab versus 0.80 ± 0.16 IU, +PLB-Ab), 14% (1.06 ± 0.13 IU, −PLB-Ab versus1.23 ± 0.15 IU, +PLB-Ab), and 64% (0.362 ± 0.09 IU, −PLB-Ab versus 1.020 ± 0.17 IU, +PLB-Ab), respectively. Thus C41L-PLB has a much smaller inhibitory effect than wt-PLB, and L37A-PLB has a much larger inhibitory effect than wt-PLB. These inhibitory effects, for both wt-PLB and L37A-PLB, are due to a shift in calcium sensitivity (an increase inK Ca) of the Ca-ATPase. As a more direct measure of PLB effects on SERCA activity, we also measured ATP hydrolysis by the reconstituted Ca-ATPase in the presence of calcium ionophore A23187 at 25 °C. With the use of the calcium ionophore, no transported Ca2+ is retained by the vesicles, allowing the ATPase to operate freely in the absence of generated Ca2+ gradients. With this method of assay, ATPase hydrolysis by the Ca2+ pump is not limited by the finite Ca2+ capacity of the reconstituted vesicles, and the permeability of the lipid vesicles themselves does not act as a confounding variable. At saturating [Ca2+] (pCa 5.4), neither wt-PLB nor the two mutants (C41L-PLB and L37A-PLB) significantly affected the ATP hydrolysis rate (Fig. 3,left panel); ATP hydrolysis rates were identical in the presence and absence of the anti-PLB antibody. At pCa 6.8 (Fig. 3, right panel) wt-PLB and L37A-PLB inhibited the ATP hydrolysis rates significantly, whereas C41L-PLB was without appreciable effect. As observed with the coexpression system (22Autry J.M. Jones L.R. J. Biol. Chem. 1997; 272: 15872-15880Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 23Kimura Y. Kurzydlowski M. Tada M. MacLennan D.H. J. Biol. Chem. 1997; 272: 15061-15064Crossref PubMed Scopus (249) Google Scholar), monomeric L37A-PLB inhibited the Ca-ATPase more effectively than pentameric wt-PLB. As observed with the Ca2+ transport assay, the inhibitory effect of co-reconstituted PLB on the ATP hydrolysis rates was prevented by the anti-PLB antibody. Thus, the co-reconstitution method with purified proteins gives results similar to those obtained with the cellular coexpression method for investigating PLB/SERCA regulatory interactions. Although the functional (inhibitory) effects of wt-PLB, C41L-PLB, and L37A-PLB obtained from ATP hydrolysis measurements are qualitatively similar to those obtained from Ca2+ uptake measurements, the overall rates of Ca2+ uptake at pCa 6.8 are significantly higher than the ATP hydrolysis rates. This could be due to the difference in lipid composition used for reconstitutions for these two different activity measurements. Also, the ATP hydrolysis rates are slightly lower in C41L-PLB/Ca-ATPase and L37A-PLB/Ca-ATPase reconstituted vesicles compared with wt-PLB/Ca-ATPase reconstituted vesicles and significantly lower compared with control (Ca-ATPase alone) vesicles. However, the Ca2+ uptake and ATP hydrolysis rates of Ca-ATPase atpCa 5.4 in the absence and presence of anti-PLB antibody are similar, indicating that the variations in the rates are not due to PLB or its mutants. The PLB-induced inhibitory effects discussed above (Figs. 2 and 3) were measured at added PLB/Ca-ATPase molar ratio of 10 in the reconstituted system. In cardiac SR, the molar ratio of PLB/Ca-ATPase is reported to be in the range of 5–8 (37Ferrington D.A. Moewe P.L. Yao Q. Bigelow D.J. Biophys. J. 1998; 74: A356Google Scholar). At an added PLB/Ca-ATPase molar ratio of 10, if the insertion of PLB in the lipid bilayers is symmetric and the Ca-ATPase is asymmetric (36.Deleted in proof.Google Scholar), then the effective PLB/Ca-ATPase molar ratio is only 5, which is on the lower side of the reported molar ratio in cardiac SR. Also, if the inhibitory species of PLB is a monomer, then increasing the molar ratio of PLB/Ca-ATPase should also increase the amount of monomer, which should reflect in the inhibitory effects of wt-PLB and its mutant, specifically C41L-PLB. To test this, we have studied the inhibitory effects of PLB and its mutants, C41L-PLB and L37A-PLB, at different molar ratios of PLB/Ca-ATPase at pCa 5.4 and 6.8. In order to ensure that the effects were not due to differences in the efficiency of incorporation, we measured directly the fraction of PLB incorporated into vesicles. PLB and its mutants, C41L-PLB and L37A-PLB, were reconstituted at added PLB/Ca-ATPase molar ratios of 5, 10, and 25 and measured the fraction of added PLB incorporated into lipid vesicles by separating free PLB from ves
Phospholamban (PLB), a 52-amino acid protein, regulates the Ca-ATPase (calcium pump) in cardiac sarcoplasmic reticulum (SR) through PLB phosphorylation mediated by β-adrenergic stimulation. The mobility of PLB on SDS−PAGE indicates a homopentamer, and it has been proposed that the pentameric structure of PLB is important for its regulatory function. However, the oligomeric structure of PLB must be determined in its native milieu, a lipid bilayer containing the Ca-ATPase. Here we have used fluorescence energy transfer (FET) to study the oligomeric structure of PLB in SDS and dioleoylphosphatidylcholine (DOPC) lipid bilayers reconstituted in the absence and presence of Ca-ATPase. PLB was labeled, specifically at Lys 3 in the cytoplasmic domain, with amine-reactive fluorescent donor/acceptor pairs. FET between donor- and acceptor-labeled subunits of PLB in SDS solution and DOPC lipid bilayers indicated the presence of PLB oligomers. The dependence of FET efficiency on the fraction of acceptor-labeled PLB in DOPC bilayers indicated that it is predominantly an oligomer having 9−11 subunits, with ∼10% of the PLB as monomer, and the distance between dyes on adjacent PLB subunits is 0.9 ± 0.1 nm. When labeled PLB was reconstituted with purified Ca-ATPase, FET indicated the depolymerization of PLB into smaller oligomers having an average of 5 subunits, with a concomitant increase in the fraction of monomer to 30−40% and a doubling of the intersubunit distance. We conclude that PLB exists primarily as an oligomer in membranes, and the Ca-ATPase affects the structure of this oligomer, but the Ca-ATPase binds preferentially to the monomer and/or small oligomers. These results suggest that the active inhibitory species of PLB is a monomer or an oligomer having fewer than 5 subunits.
Abstract TLR agonists have been used in treating diseases including infections, cancer and as vaccine adjuvants. Aldara™ (imiquimod 5% cream) is the first TLR7 agonist approved in the US, and is indicated for the treatment of genital warts caused by human papillomavirus (HPV). Aldara was not well-tolerated when delivered vaginally; therefore a 2nd generation analog, 851, that retained the ability to stimulate cytokine production upon vaginal application was selected for development to treat high-risk cervical HPV infection. In human PBMC and purified plasmacytoid DC cultures, 851 stimulated a >10 fold increase in type I IFN, and indirectly activated human NK cells to express CD69 (8-fold) at concentrations of ≥10μM. In the rat, intravaginal application of 851 gel (0.01% and 0.1% w/v) led to local tissue production of TNF-α and MCP-1 without evidence of systemic cytokine induction. Increases in vaginal levels of TNF-α (9-fold) and MCP-1 (81-fold) were seen with the top strength of 851 tested (0.1%). Vaginal application of 851 results in the influx of NK cells, T cells and macrophages and an efflux of DC from the vaginal tissue. The ability of 851 to induce local cytokine production leading to influx and efflux of various immune cell populations could result in elimination of virally-infected cells from the cervix and vaginal tract including cells infected with high-risk HPV that are known to lead to cervical cancer.
