Interaction of nucleotides and cations with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate

• Published in: The Journal of biological chemistry Vol. 258; no. 17; pp. 10433 - 10438
• Main Authors: Arav, R, Aderem, A A, Berman, M C
• Format: Journal Article
• Language: English
• Published: Bethesda, MD Elsevier Inc 10-09-1983; American Society for Biochemistry and Molecular Biology
• Subjects: Analytical, structural and metabolic biochemistry; Anilino Naphthalenesulfonates - metabolism; Animals; Biological and medical sciences; Ca(2+) Mg(2+)-ATPase; Calcium - metabolism; Calcium-Transporting ATPases - metabolism; Cations, Divalent - metabolism; Egtazic Acid - pharmacology; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; Hydrolases; Kinetics; Nucleotides - metabolism; Rabbits; Sarcoplasmic Reticulum - enzymology; Spectrometry, Fluorescence; Valinomycin - pharmacology; Vanadium - pharmacology; Enzyme; Fluorescent labelling; Sarcoplasmic reticulum; Ca,Mg-ATPase; Fluorescence; Nucleotide; Molecular interaction
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(17)44475-9

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 Kd 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, Kd = 1.0 X 10(-5) M, nH = 0.80; for ADP, Kd = 1.2 X 10(-5) M, nH = 0.89; and for Ca2+, Kd = 3.4 X 10(-7) M, nH = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl-beta, gamma-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 states of the (Ca2+, Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence (DuPont, Y. (1976) Biochem, Biophys. Res. Commun. 71, 544-550).