Conformational change accompanying formation of oligomycin-induced Na(+)-bound forms and their conversion to ADP-sensitive phosphoenzymes in Na+,K(+)-ATPase.
Oligomycin reduced the fluorescence intensity of an N-(p-(2-benzimidazoly)phenyl) maleimide (BIPM) probe at Cys-964 of the alpha-chain of pig kidney Na+,K(+)-ATPase with increase in the concentration of Na+ with a Hill coefficient of nh = 0.77 with Kh = 231 mM. The maximum fluorescence decrease was around 80% of the value observed after accumulation of ADP-sensitive phosphoenzyme (E1P) in the presence of 2 M Na+. The addition of Mg2+ and ATP with Na+ or choline chloride to give the same final ligand concentration to the Na(+)-enzyme-oligomycin complex formed with 16 mM Na+ + 1,984 mM choline chloride or 2 M Na+ induced rapid phosphorylation (20 or 21/s) and slower fluorescence decrease (12.1 +/- 1.2 or 10.1 +/- 3.2/s). These additions to the Na(+)-enzyme complex formed under the former or the latter conditions induced slow phosphorylation (13/s) prior to a much slower fluorescence decrease (3.4 +/- 0.3 or 8.6 +/- 0.7/s). The addition of Ca2+ and ATP to these enzyme complexes induced rapid fluorescence changes (21-11/s) followed by one order of magnitude slower rates of phosphorylation (1.5-1.3 s). These data suggest that the decrease in BIPM fluorescence induced by ATP with Ca2+ or with Mg2+, reflects the change of the Na+ binding state before or after the formation of E1P, respectively.
Taniguchi
K
K
Department of Chemistry, Faculty of Science, Hokkaido University.
Sasaki
T
T
Shinoguchi
E
E
Kamo
Y
Y
Ito
E
E
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology; D005456:Fluorescent Dyes; D006868:Hydrolysis; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008301:Maleimides; D009840:Oligomycins / Q000494:pharmacology*; D010042:Ouabain / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D011487:Protein Conformation; D012965:Sodium Chloride / Q000378:metabolism*; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013552:Swine
D005456:Fluorescent Dyes; D008301:Maleimides; D009840:Oligomycins; D012965:Sodium Chloride; C000245:N-(4-(2-benzimidazolyl)phenyl)maleimide; D010042:Ouabain; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium; D002118:Calcium