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We have measured resonance energy transfer between two donor-acceptor pairs localized on different domains of the Ca-ATPase of sarcoplasmic reticulum in order to determine whether changes in tertiary structure accompany active calcium transport. Energy transfer was determined from both steady state intensities and time-resolved lifetimes of 5-(2-((acety1)- amino)ethyDaminonaphthalene-l-sulfonic acid (IAEDANS), specifically bound to the B tryptic fragment, using two acceptors: (1) fluorescein 5'-isothiocyanate (FITC), covalently bound at the nucleotide site, also on the B fragment, and (2) 4-dimethylaminophenylazopheny1-4'- maleimide (DABMI), bound on the Al subfragment. Neither binding of calcium to the high affinity sites nor phosphorylation by inorganic phosphate is accompanied by detectable changes in the distance between IAEDANS and FITC, suggesting that the B fragment does not undergo any large-scale (>1 A) physical distortion under these conditions. On the other hand, measurements of energy transfer from IAEDANS to the acceptor DABMI, on the Al subfragment, demonstrate that phosphorylation with inorganic phosphate or addition of pM VO4 results in increased energy transfer, that is reversible with subsequent addition of calcium. Addition of calcium to the nonphosphorylated enzyme results in no detectable change in energy transfer. The presence of the detergent lysolecithin prevents the phosphate dependent increase in fluorescence energy transfer, suggesting that protein-protein interactions may contribute to the observed change in energy transfer. A direct relationship between an increased degree of protein-protein interactions and phosphoenzyme formation is confirmed by investigations using a reconstituted preparation containing a mixed popu Lation of Ca-ATPase polypeptide chains labeled either with IAEDANS or with DABMI. These results suggest a phosphorylation dependent change in either the affinity or orientation of Ca-ATPase polypeptide chains with respect to one another.
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