Functional characterization of lanthanide binding sites in the sarcoplasmic reticulum Ca(2+)-ATPase: do lanthanide ions bind to the calcium transport site?
Gd3+ binding sites on the purified Ca(2+)-ATPase of sarcoplasmic reticulum were characterized at 2 and 6 degrees C and pH 7.0 under conditions in which 45Ca2+ and 54Mn2+ specifically labeled the calcium transport site and the catalytic site of the enzyme, respectively. We detected several classes of Gd3+ binding sites that affected enzyme function: (a) Gd3+ exchanged with 54Mn2+ of the 54MnATP complex bound at the catalytic site. This permitted slow phosphorylation of the enzyme when two Ca2+ ions were bound at the transport site. The Gd3+ ion bound at the catalytic site inhibited decomposition of the ADP-sensitive phosphoenzyme. (b) High-affinity binding of Gd3+ to site(s) distinct from both the transport site and the catalytic site inhibited the decomposition of the ADP-sensitive phosphoenzyme. (c) Gd3+ enhanced 4-nitro-2,1,3-benzoxadiazole (NBD) fluorescence in NBD-modified enzyme by probably binding to the Mg2+ site that is distinct from both the transport site and the catalytic site. (d) Gd3+ inhibited high-affinity binding of 45Ca2+ to the transport site not by directly competing with Ca2+ for the transport site but by occupying site(s) other than the transport site. This conclusion was based mainly on the result of kinetic analysis of displacement of the enzyme-bound 45Ca2+ ions by Gd3+ and vice versa, and the inability of Gd3+ to phosphorylate the enzyme under conditions in which GdATP served as a substrate. These results strongly suggest that Ln3+ ions cannot be used as probes to structurally and functionally characterize the calcium transport site on the Ca(2+)-ATPase.
Ogurusu
T
T
Department of Molecular Physiology, National Cardiovascular Center Research Institute, Osaka, Japan.
Wakabayashi
S
S
Shigekawa
M
M
D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D001692:Biological Transport / Q000187:drug effects; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002384:Catalysis / Q000187:drug effects; D005682:Gadolinium / Q000494:pharmacology; D007700:Kinetics; D008345:Manganese / Q000494:pharmacology; D008674:Metals, Rare Earth / Q000494:pharmacology*; D010750:Phosphoproteins / Q000187:drug effects; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000201:enzymology*
D008674:Metals, Rare Earth; D010750:Phosphoproteins; D008345:Manganese; D005682:Gadolinium; D000252:Calcium-Transporting ATPases; D002118:Calcium