title: Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca(2+)-ATPase.
abstract: Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca(2+)-ATPase in response to beta-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(2+)-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(2+)-ATPase but had no effect on Vmax. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca(2+)-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 approximately 3:1, PLB/Ca(2+)-ATPase. We also investigated peptides corresponding to the two main domains of PLB. The membrane-spanning domain, PLB26-52, appeared to uncouple ATPase hydrolysis from calcium transport, even though the permeability of the reconstituted vesicles was not altered. The cytoplasmic peptide, PLB1-31, had little effect, even at a 300:1 molar excess over Ca(2+)-ATPase.
authors.affiliation: Department of Molecular Physiology and Biologial Physics, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
authors.forename: L G
authors.initials: LG
authors.lastname: Reddy
authors.forename: L R
authors.initials: LR
authors.lastname: Jones
authors.forename: S E
authors.initials: SE
authors.lastname: Cala
authors.forename: J J
authors.initials: JJ
authors.lastname: O'Brian
authors.forename: S A
authors.initials: SA
authors.lastname: Tatulian
authors.forename: D L
authors.initials: DL
authors.lastname: Stokes
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000502:physiology*; D000252:Calcium-Transporting ATPases / Q000502:physiology*; D018482:Muscle, Skeletal / Q000201:enzymology*; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D018411:Spodoptera; D000099211:Phospholamban
chemical_list: D002118:Calcium; D002135:Calcium-Binding Proteins; D000252:Calcium-Transporting ATPases; D011994:Recombinant Proteins; D000099211:Phospholamban