valinomycin and 1-anilino-8-naphthalenesulfonate

1-anilino-8-naphtalenesulfonate (ANS) is a hydrophobic dipole previously used to demonstrate that the proton for potassium exchange by the gastric HK-ATPase is electroneutral. In this paper, we demonstrate that ANS binds to gastric membranes and probes conformational changes of the HK-ATPase independently of any active H for K exchange. Conformational changes require the presence of potassium-valinomycin and are not triggered by sodium. Potassium effect is enhanced by ATP, in the presence and in the absence of magnesium and, by ADP, in the presence of magnesium. Labeling of the pig HK-ATPase K518 by fluorescein-5-isothiocyanate inhibits the enzyme activity and knocks out the ATP effect on ANS fluorescence. Scherring 28080 and the monoclonal antibody 95-111, two competitive inhibitors of K-activated ATPase dephosphorylation, do not modify K-effect on ANS fluorescence but inhibit ATP effects. This supports that ANS does not probe K-site between the H1-H2 loop. Treatment of gastric membranes with trypsin does not inhibit the ANS response to potassium but does inhibit the response to ATP. This suggests that the ATP site inducing the ANS response is cytoplasmic and the potassium site is intramembranous. Titration reveals that one mole of ANS interacts with one mole of ATPase. We suggest that ANS probes a hydrophobic potassium site of gastric ATPase and that addition of ATP and ADP-Mg embed that site.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Anilino Naphthalenesulfonates; Animals; Fluorescent Dyes; H(+)-K(+)-Exchanging ATPase; Ionophores; Models, Molecular; Potassium; Protein Folding; Stomach; Valinomycin

Sperm from trout, like other sperm, are immotile in the seminal tract and initiate motility upon dilution into an appropriate fertilizing environment. Trout sperm motility is inhibited by high extracellular [K+] and can be activated by dilution of extracellular [K+]. Activation of trout sperm by the dilution of extracellular [K+] suggests regulation by membrane potential. Using the membrane potential-sensitive fluorescent dye 3,3'-dipropylthiocarbocyanine iodide (diS-C3-(5)) we directly measured the K+ contribution to the membrane potential. Manipulating the membrane potential with Cs+ and the ionophore valinomycin can override K+ regulation. We show that trout sperm can also be activated in the presence of inhibitory [K+] by the addition of divalent cations. Activation by divalent cations is explained by the cations' ability to mask membrane surface potential and thus alter the potential sensed by membrane voltage sensors. Using the surface potential-sensitive dye, 1-anilino-8-naphthosulfonate (ANS), we directly measure the divalent cations' ability to mask surface potential. We propose a model where membrane hyperpolarization is the trigger that initiates the cascade of events leading to trout sperm activation. An increase in intracellular pH has been suggested to be a conserved step in the activation of sperm motility. We show that increasing intracellular pH by procedures that activate sea urchin and mammalian sperm does not activate trout sperm. In contrast, there is a decrease in intracellular pH upon activation of trout sperm motility. Artificially decreasing intracellular pH is not sufficient for activation of motility in trout sperm in an inhibitory [K+]. Thus, unlike some other sperm, changes in intracellular pH do not regulate trout sperm motility.

Topics: Anilino Naphthalenesulfonates; Animals; Benzothiazoles; Carbocyanines; Cesium; Fluoresceins; Hydrogen-Ion Concentration; Magnesium; Male; Membrane Potentials; Models, Biological; Potassium; Sperm Motility; Spermatozoa; Trout; Valinomycin

Lipid vesicles have been utilized to study the interactions of diphtheria toxin (DT) with membranes. The assay for DT ion channel formation was fluorescence-detected membrane potential depolarization of vesicles in which valinomycin-induced potassium diffusion gradients had been generated. The following requirements for ion channel formation have been identified: (1) acid pH (less than 5); (2) trans-negative membrane potentials (35-fold increase in channel-forming activity from -6 mV to -59 mV); and (3) negatively charged phospholipid headgroups (about 100-fold more activity using vesicles formed from asolectin compared to soybean phosphatidylcholine). Concentration dependence plots of toxin activity showed a linear response with logarithmic slopes of nearly one for each lipid composition. These results show a close parallel to those obtained previously with planar lipid bilayers and thus provide guidelines for conditions which facilitate functional insertion of the toxin into vesicles.

Topics: Anilino Naphthalenesulfonates; Diffusion; Diphtheria Toxin; Fluorescent Dyes; Hydrogen-Ion Concentration; Ion Channels; Liposomes; Membrane Potentials; Phosphatidylcholines; Phospholipids; Potassium; Spectrometry, Fluorescence; Valinomycin

It has been found in the brush border membrane vesicles from porcine small intestine that valinomycin causes vesicle aggregation and that Ca2+-induced aggregation of the vesicles is saturably stimulated in the presence of valinomycin. The apparent half-maximal concentration of valinomycin required to enhance the Ca2+ effect on the membrane aggregation is approximately 40 microM. Results of a fluorometric study using 1-anilino-8-naphthalene sulfonate (ANS) showed that the addition of valinomycin to the membranes induces slight decreases in the ANS-binding affinities for the membranes in the presence and absence of Ca2+. On the other hand, measurements of the incorporation rate of 1,6-diphenyl-1,3,5-hexatriene (DPH) into the vesicles and of anisotropies of DPH-labeled membranes suggested that the lipid organization of the membranes was not altered upon the addition of valinomycin. From these results, it was suggested that the valinomycin effect on the membrane aggregation is mainly related to the nature of the membrane surface charge.

Topics: Anilino Naphthalenesulfonates; Animals; Fluorescent Dyes; Intestine, Small; Kinetics; Microvilli; Spectrometry, Fluorescence; Swine; Valinomycin

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).

Topics: Anilino Naphthalenesulfonates; Animals; Ca(2+) Mg(2+)-ATPase; Calcium; Calcium-Transporting ATPases; Cations, Divalent; Egtazic Acid; Kinetics; Nucleotides; Rabbits; Sarcoplasmic Reticulum; Spectrometry, Fluorescence; Valinomycin; Vanadium