title: Detection of conformational changes in chloroplast coupling factor 1 by 8-anilino-1-naphthalene-sulphonate fluorescence changes.
abstract: Chloroplast coupling factor 1 (CF1) contains a high-affinity binding site for 8-anilino-1-napthalene sulphonate (ANS,Kd = 5-6 microM). The binding of ANS to the enzyme is associated with a fluorescence enhancement and a blue-shift in the emission spectrum. ANS only slightly inhibits ATP hydrolysis by CF1. Adenine nucleotides and inorganic phosphate induce a fast ANS fluorescence quenching of about 50% which is due to a decrease in the affinity of the enzyme for ANS (Kd increases from 6 microM to 22 microM) and in the fluorescence quantum yield of the bound probe (by 33%) but not in the number of ANS sites (n = 1). Conversely, Mg and Ca ions induce a fluorescence enhancement of bound ANS. Inactivation of the enzyme enhances ANS fluorescence, eliminates the response to adenine nucleotides and inorganic phosphate but increases the response to divalent metals. The affinity of latent CF1 for ADP (Kd = 12 microM) is considerably higher than for ATP (Kd = 95 microM) in buffer containing EDTA. The Kd for inorganic phosphate is 140 microM. Mg increases the apparent affinity for ATP (Kd = 28 microM) but not for ADP or Pi. Binding of ATP to the tight-sites does not inhibit the ADP or Pi-induced fluorescence quenching but decreases the affinity for ADP (Kd = 34 microM) and for inorganic phosphate (Kd = 320 microM). These results suggest that the ADP and phosphate binding sites are different but not independent from the tight sites. Activation of a Mg-specific ATPase in CF1 by octyl glucoside decreases the affinity for ADP and inorganic phosphate by about threefold but increases the affinity for ATP. ATPase activation of CF1 also increases the Ki for ADP inhibition of ATP hydrolysis. ATPase activation also influences the ANS responses to Ca and Mg. Ca-ATPase activation increases the fluorescence enhancement and the apparent affinity for Ca whereas Mg-ATPase activation specifically increases the Mg-induced fluorescence enhancement. The fluorescence of CF1-bound ANS is enhanced by Dio-9 and quenched by phloridzin, quercetin, Nbf-Cl and FITC. Nbf-Cl and FITC completely inhibit the ADP-induced fluorescence quenching whereas Dio-9 inhibits the Mg-induced fluorescence enhancement. ANS does not relieve the quercetin or phloridzin inhibition of ATP hydrolysis indicating that these inhibitors do not compete with ANS for a common binding site. ANS may be used, therefore, as a sensitive probe to detect conformational changes in CF1 in response to activation or inactivation and to binding of substrates and of inhibitors.
mesh_terms: D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000817:Anilino Naphthalenesulfonates / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D055598:Chemical Phenomena; D002621:Chemistry; D008024:Ligands / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D010710:Phosphates / Q000494:pharmacology; D011485:Protein Binding; D011487:Protein Conformation; D006180:Proton-Translocating ATPases / Q000302:isolation & purification*; D013050:Spectrometry, Fluorescence
chemical_list: D000817:Anilino Naphthalenesulfonates; D008024:Ligands; D010710:Phosphates; D000244:Adenosine Diphosphate; C027132:1-anilino-8-naphthalenesulfonate; D000255:Adenosine Triphosphate; D006180:Proton-Translocating ATPases; D008274:Magnesium; D002118:Calcium