Eosin, a fluorescent marker for the high-affinity ATP site of (K++H+)-ATPase

• Published in: Biochimica et biophysica acta Vol. 858; no. 2; pp. 254 - 262
• Main Authors: HELMICH DE JONG, M. L, VAN DUYNHOVEN, J. P. M, SCHUURMANS STEKHOVEN, F. M. A. H, DE PONT, J. J. H. H. M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier 26.06.1986; North-Holland
• Subjects: Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Animals; Binding Sites; Binding, Competitive; Biological and medical sciences; Choline - pharmacology; Eosine Yellowish-(YS) - metabolism; Fluorescent Dyes; Fundamental and applied biological sciences. Psychology; Gastric Mucosa - enzymology; H(+)-K(+)-Exchanging ATPase; Interactions. Associations; Intermolecular phenomena; Magnesium - pharmacology; Molecular biophysics; Osmolar Concentration; Potassium - pharmacology; Protein Conformation; Spectrometry, Fluorescence; Swine; Stomach; Fluorescent tracer; Enzyme; Mucosa; Binding site; Pig; Vertebrata; Mammalia; Molecular association; Artiodactyla; ATP; Conformation induction; Ungulata
• ISSN: 0006-3002; 1878-2434
• DOI: 10.1016/0005-2736(86)90330-5

Eosin has been used as a fluorescent probe for studying conformational states in (K+ + H+)-ATPase. The eosin fluorescence level is increased by Mg2+ (K0.5 = 0.2 mM). This increase is counteracted by K+ (I0.5 = 1.3 mM) and choline (I0.5 = 17.2 mM) and by ATP. Binding studies with eosin indicate that the increase and decrease in fluorescence is due to changes in binding of eosin to the enzyme. The Mg2+-induced specific binding has a Kd of 0.7 microM and a maximal capacity of 3.5 nmol per mg enzyme, which is equivalent to 2.5 site per phosphorylation site. These experiments and the fact that eosin competitively inhibits (K+ + H+)-ATPase towards ATP, suggest that eosin binds to ATP binding sites.