Lg. Reddy et al., FUNCTIONAL RECONSTITUTION OF RECOMBINANT PHOSPHOLAMBAN WITH RABBIT SKELETAL CA2-ATPASE(), The Journal of biological chemistry, 270(16), 1995, pp. 9390-9397
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 P-adrenergic stimulation. We have used the
baculovirus expression system in Sf21 cells to express milligram quant
ities of wild-type PLB. After purification by antibody affinity chroma
tography, the function of this recombinant PLB was tested by reconstit
ution 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 V-max. 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 monoclona
l antibody to PLB, which mimics the physiological effects of PLB phosp
horylation. Maximal PLB regulatory effects occurred at a molar stoichi
ometry of similar to 3:1, PLB/Ca2+-ATPase. We also investigated peptid
es corresponding to the two main domains of PLB. The membrane-spanning
domain, PLB(26-52), appeared to uncouple ATPase hydrolysis from calci
um transport, even though the permeability of the reconstituted vesicl
es was not altered. The cytoplasmic peptide, PLB(1-31), had little eff
ect, even at a 300:1 molar excess over Ca2+-ATPase.