TY - JOUR
T1 - Interaction of nucleotides and cations with the (Ca2+,Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate
AU - Arav, R.
AU - Aderem, A. A.
AU - Berman, M. C.
N1 - Copyright:
Copyright 2013 Elsevier B.V., All rights reserved.
PY - 1983
Y1 - 1983
N2 - The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+,Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent K(d) of 3.8 x 10-5 M. The binding of ANS- had no effect on Ca2+ transport or Ca2--dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, K(d) = 1.0 x 10-5 M, n(H) = 0.80; for ADP, K(d) = 1.2 x 10-5 M, n(H) = 0.89; and for Ca2+, K(d) = 3.4 x 10-7 M, n(H) = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl(-β,γ-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS- with half-maximal effect at 4 x 10-5 M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+,Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational rates of the (Ca2+,Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence.
AB - The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+,Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent K(d) of 3.8 x 10-5 M. The binding of ANS- had no effect on Ca2+ transport or Ca2--dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, K(d) = 1.0 x 10-5 M, n(H) = 0.80; for ADP, K(d) = 1.2 x 10-5 M, n(H) = 0.89; and for Ca2+, K(d) = 3.4 x 10-7 M, n(H) = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl(-β,γ-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS- with half-maximal effect at 4 x 10-5 M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+,Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational rates of the (Ca2+,Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence.
UR - http://www.scopus.com/inward/record.url?scp=0021062690&partnerID=8YFLogxK
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C2 - 6136508
AN - SCOPUS:0021062690
SN - 0021-9258
VL - 258
SP - 10433
EP - 10438
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 17
ER -