A. Norregaard et al., CHIMERIC CA2-ATPASE()NA+,K+-ATPASE MOLECULES - THEIR PHOSPHOENZYME INTERMEDIATES AND SENSITIVITY TO CA2+ AND THAPSIGARGIN/, FEBS letters, 336(2), 1993, pp. 248-254
Chimeric molecules consisting of parts from the sarcoplasmic reticulum
Ca2+-ATPase and the Na+,K+-ATPase were expressed in COS-1 cells and a
nalysed functionally. One chimera, in which most of the central cytopl
asmic loop was derived from the Na+,K+-ATPase, while the transmembrane
segments and the minor cytoplasmic loop came from the Ca2+-ATPase, wa
s able to occlude Ca2+ and to be phosphorylated from ATP with normal a
pparent affinity for Ca2+ and ATP. This chimera also displayed normal
sensitivity to thapsigargin, but was unable to undergo the transition
from ADP-sensitive to ADP-insensitive phosphoenzyme and to transport C
a2+. The other chimera, which consisted of the NH2-terminal two-thirds
of Na+,K+-ATPase and the COOH-terminal one-third of Ca2+-ATPase, was
unable to phosphorylate from ATP, but phosphorylated from inorganic ph
osphate in a Ca2+-inhibitable and thapsigargin-insensitive reaction. T
hese results can be explained in terms of a structural model in which
the non-conserved residues in the central cytoplasmic domain of the Ca
2+-ATPase are without major importance for the binding and occlusion o
f Ca2+, but are involved in the E1P --> E2P conformational changes of
the phosphoenzyme, whereas residues in transmembrane segments on both
sides of the central cytoplasmic domain are involved in formation of t
he Ca2+-binding sites. The data moreover show that thapsigargin sensit
ivity is dependent on residues in the NH2-terminal one-third of the Ca
2+-ATPase molecule.