Distances between the functional sites of the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum.
Luminescence energy transfer measurements have been used to determine the distances between the two high affinity Ca2+ binding-transport sites of the (Ca2+ + Mg2+)-ATPase of skeletal muscle sarcoplasmic reticulum. The lanthanide Tb3+ situated at one high affinity Ca2+ site was used as the transfer donor, and acceptors at the other Ca2+ site were the lanthanides Nd3+, Pr3+, Ho3+, or Er3+. Terbium bound to the enzyme was excited directly with a pulsed dye laser. Analysis of the changes in the terbium luminescence lifetime due to the presence of the acceptor indicates that the distance between the Ca2+ sites is 10.7 A. The distance between the Ca2+ sites and the nucleotide-binding catalytic site was determined using Tb3+ at the Ca2+ sites and either trinitrophenyl nucleotides (TNP-N) or fluorescein 5-isothiocyanate (FITC) in the catalytic site as energy acceptors. The R0 values for the Tb-acceptor pairs are approximately 30 and approximately 40 A for TNP-N and FITC, respectively. The distance between Tb3+ at the Ca2+ sites and TNP-ATP at the nucleotide site is approximately 35 A and that between the Ca2+ sites and the FITC labeling site is approximately 47 A. Considerations of the molecular dimensions of the ATPase polypeptide indicate that while the two Ca2+ sites are close to each other, the Ca2+ sites and the nucleotide site are quite remote in the three-dimensional structure of the enzyme.
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D000818:Animals; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007700:Kinetics; D008163:Luminescent Measurements; D009132:Muscles / Q000201:enzymology; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013861:Thiocyanates
D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D016650:Fluorescein-5-isothiocyanate