A K(+)-competitive fluorescent inhibitor of the H,K-ATPase

• Published in: The Journal of biological chemistry Vol. 266; no. 19; pp. 12395 - 12401
• Main Authors: RABON, E, SACHS, G, BASSILIAN, S, LEACH, C, KEELING, D
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 05.07.1991
• Subjects: Adenosine Triphosphatases - antagonists & inhibitors; Adenosine Triphosphatases - metabolism; Adenosine Triphosphate - metabolism; Aminoquinolines - metabolism; Analytical, structural and metabolic biochemistry; Binding, Competitive; Biological and medical sciences; Biological Transport; Catalysis; Enzymes and enzyme inhibitors; Fluorescent Dyes - metabolism; Fundamental and applied biological sciences. Psychology; H(+)-K(+)-Exchanging ATPase; Hydrogen - metabolism; Hydrolases; Nigericin - pharmacology; Phosphorylation; Potassium - metabolism; Stoichiometry; Enzyme; H; Ion transport; Molecular interaction; K; Competitive inhibition; Microsome; transporting ATPase; Enzymatic activity; Inhibition; Fluorescent compound; Conformational transition
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)98910-6

The interactions of a novel fluorescent compound, 1-(2-methylphenyl)-4-methylamino-6-methyl-2,3-dihydropyrrolo[3,2-c ]quinoline (MDPQ) with the gastric H,K-ATPase were determined. MDPQ was shown to inhibit the H,K-ATPase and its associated K(+)-phosphatase competitively with K+, with Ki values of 0.22 and 0.65 microM, respectively. It also inhibited H+ transport with an IC50 of 0.29 microM, but at a concentration of 3.5 microM, reduced the steady-state level of phosphoenzyme by only 28%. The fluorescence of the inhibitor increased upon binding to the enzyme. 70% of this increment was quenched by K+, independently of Mg2+. The binding of MgATP to a high affinity site (K0.5(ATP) less than 1 microM) markedly increased the fluorescence due to the formation of an inhibitor-phosphoenzyme complex saturating with a K0.5(MDPQ) of 0.94 microM. The K(+)-dependent fluorescent quench (K0.5(K+) = 1.8 mM) required the ionophore, nigericin, indicating that K+ and MDPQ were competing at an extracytosolic site on the enzyme. Formation also of an enzyme-vanadyl-inhibitor complex was shown by the fact that Mg2+ plus vanadate enhanced MDPQ fluorescence in the absence of MgATP and decreased fluorescence in the presence of MgATP. The minimal stoichiometry of bound MDPQ determined by fluorescence titrations in the presence of MgATP was 1.4 mol/mol phosphoenzyme. The data suggest that this compound can serve as a probe of conformation at an extracytosolic site of the H,K-ATPase.