Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPaSe

Laxma G. Reddy, Larry Jones, Steven E. Cala, Jeffrey J. O'Brian, Suren A. Tatulian, David L. Stokes

Research output: Contribution to journalArticle

96 Citations (Scopus)

Abstract

Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca2+-ATPase in response to β-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 Ca2+-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 Ca2+-ATPase but had no effect on Vmax. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca2+-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/Ca2+-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 Ca2+-ATPaSe.

Original languageEnglish
Pages (from-to)9390-9397
Number of pages8
JournalJournal of Biological Chemistry
Volume270
Issue number16
StatePublished - Apr 21 1995

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Rabbits
Calcium-Transporting ATPases
Sarcoplasmic Reticulum
Calcium
Adenosine Triphosphatases
phospholamban
Affinity chromatography
Sf9 Cells
Peptides
Phosphorylation
Antibody Affinity
Baculoviridae
Affinity Chromatography
Stoichiometry
Adrenergic Agents
Purification
Canidae
Hydrolysis
Permeability
Monoclonal Antibodies

ASJC Scopus subject areas

  • Biochemistry

Cite this

Reddy, L. G., Jones, L., Cala, S. E., O'Brian, J. J., Tatulian, S. A., & Stokes, D. L. (1995). Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPaSe. Journal of Biological Chemistry, 270(16), 9390-9397.

Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPaSe. / Reddy, Laxma G.; Jones, Larry; Cala, Steven E.; O'Brian, Jeffrey J.; Tatulian, Suren A.; Stokes, David L.

In: Journal of Biological Chemistry, Vol. 270, No. 16, 21.04.1995, p. 9390-9397.

Research output: Contribution to journalArticle

Reddy, LG, Jones, L, Cala, SE, O'Brian, JJ, Tatulian, SA & Stokes, DL 1995, 'Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPaSe', Journal of Biological Chemistry, vol. 270, no. 16, pp. 9390-9397.
Reddy LG, Jones L, Cala SE, O'Brian JJ, Tatulian SA, Stokes DL. Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPaSe. Journal of Biological Chemistry. 1995 Apr 21;270(16):9390-9397.
Reddy, Laxma G. ; Jones, Larry ; Cala, Steven E. ; O'Brian, Jeffrey J. ; Tatulian, Suren A. ; Stokes, David L. / Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca2+-ATPaSe. In: Journal of Biological Chemistry. 1995 ; Vol. 270, No. 16. pp. 9390-9397.
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