RESUMEN
In the paper, we present a qualitative analysis of the dual-pulse phase optical time domain reflectometry (phase-OTDR) response to uniform and nonuniform propagating fiber strain. It is found that on average over all realizations of scattering centers the response of the dual-pulse phase-OTDR is linear with respect to an external perturbation. Meanwhile, individual responses contain random phase jumps, which are an intrinsic property of phase-OTDR. These jumps are the result of nonlinear responses of the scattering fiber segments and arise due to interference of random backscattered fields varying in time. Two types of phase jumps are considered: π jumps and 2π jumps; the first type is caused by the fading in phase-OTDR spatial channel, while the second type occurs when a nonuniform perturbation propagates along the fiber. The origin of the phase jumps is explained by considering the simulated response on the complex plane. It is shown that the distribution of 2π jumps can be well described by the Gaussian probability mass function (PMF), provided the number of 2π jumps is large. The conducted experiments on the registration of uniform and nonuniform fiber strain confirm the presence of the jumps in the phase-OTDR response.
RESUMEN
A distributed acoustic sensor (a phase optical time-domain reflectometer) configuration with a low noise level in the hertz and sub-hertz frequency ranges is proposed. The sensor scheme uses a Mach-Zehnder interferometer to generate a dual-pulse probe signal and implements the frequency stabilization of a laser source using the same interferometer as a frequency etalon. The scheme simultaneously provides a low noise level owing to the compensation of the optical path difference of interfering backscattered fields and low drift of the output signal. It has been shown experimentally that the stabilization of the laser frequency provides up to 35 dB signal/noise gain in the sub-hertz frequencies, which are of interest for seismology. The applicability of the proposed scheme is demonstrated experimentally by teleseismic earthquakes recorded by a fiber-optic cable deployed on the seabed of the Black Sea.
RESUMEN
The possibility of distributed wide-range strain and temperature measurements in a 100 km long optical fiber using tunable-wavelength low-coherence optical time-domain reflectometer (OTDR) is demonstrated. The specified distance range is provided by employing two narrowband microelectromechanical system (MEMS) spectral filters tuned synchronously as well as by taking advantage of Raman amplification and amplification by remotely pumped erbium-doped fiber segments built into the fiber under test. With the time of a single measurement of 10 min and the spatial resolution of about 1 m, the measurement range reached 1000 µÉ in strain units, which is equivalent to the temperature range of 110°C.
RESUMEN
Acute lung injury (ALI) is one of the leading causes of death in sepsis. Endothelial inflammation and dysfunction play a prominent role in development of ALI. Glycolysis is the predominant bioenergetic pathway for endothelial cells (ECs). However, the role of EC glycolysis in ALI of sepsis remains unclear. Here we show that both the expression and activity of PFKFB3, a key glycolytic activator, were markedly increased in lipopolysaccharide (LPS)-treated human pulmonary arterial ECs (HPAECs) in vitro and in lung ECs of mice challenged with LPS in vivo. PFKFB3 knockdown significantly reduced LPS-enhanced glycolysis in HPAECs. Compared with LPS-challenged wild-type mice, endothelial-specific Pfkfb3 knockout (Pfkfb3ΔVEC) mice exhibited reduced endothelium permeability, lower pulmonary edema, and higher survival rate. This was accompanied by decreased expression of intracellular adhesion molecule-1 (Icam-1) and vascular cell adhesion molecule 1 (Vcam-1), as well as decreased neutrophil and macrophage infiltration to the lung. Consistently, PFKFB3 silencing or PFKFB3 inhibition in HPAECs and human pulmonary microvascular ECs (HPMVECs) significantly downregulated LPS-induced expression of ICAM-1 and VCAM-1, and monocyte adhesion to human pulmonary ECs. In contrast, adenovirus-mediated PFKFB3 overexpression upregulated ICAM-1 and VCAM-1 expression in HPAECs. Mechanistically, PFKFB3 silencing suppressed LPS-induced nuclear translocation of nuclear factor κB (NF-κB)-p65, and NF-κB inhibitors abrogated PFKFB3-induced expression of ICAM-1 and VCAM-1. Finally, administration of the PFKFB3 inhibitor 3PO also reduced the inflammatory response of vascular endothelium and protected mice from LPS-induced ALI. Overall, these ï¬ndings suggest that targeting PFKFB3-mediated EC glycolysis is an efï¬cient therapeutic strategy for ALI in sepsis.
Asunto(s)
Lesión Pulmonar Aguda/metabolismo , Células Endoteliales/metabolismo , Endotoxemia/inducido químicamente , Endotoxemia/metabolismo , Lipopolisacáridos/farmacología , Fosfofructoquinasa-2/metabolismo , Animales , Modelos Animales de Enfermedad , Endotelio Vascular/metabolismo , Glucólisis/fisiología , Humanos , Inflamación/metabolismo , Molécula 1 de Adhesión Intercelular/metabolismo , Pulmón/metabolismo , Ratones , Monocitos/metabolismo , FN-kappa B/metabolismo , Oxitocina/metabolismo , Edema Pulmonar/metabolismo , Sepsis/metabolismo , Transducción de Señal/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
OBJECTIVE AND DESIGN: The peptide from C-terminal domain of MCP-1 (Ingramon) has been shown to inhibit monocyte migration and possess anti-inflammatory activity in animal models of inflammation and post-angioplasty restenosis. Here, we investigate the effect of Ingramon treatment on blood levels of acute-phase reactants and chemokines in patients after coronary stenting and the mechanisms of Ingramon anti-inflammatory activity. SUBJECTS: Eighty-seven patients with ischemic heart disease (IHD) who faced the necessity of coronary angiography (CA) were enrolled. In 67 patients, one-stage coronary stenting was performed; 33 of them were treated with Ingramon in addition to standard therapy. Twenty patients underwent CA only. METHODS: High-sensitivity C-reactive protein (hsCRP) and fibrinogen blood levels were detected routinely. The chemokine concentration in plasma was measured by enzyme-linked immunosorbent assay (ELISA) or cytometric bead array-based immunoassay. Intracellular Ca(2+) levels and cell surface integrin exposure were assayed by flow cytometry. MCP-1 dimerization was studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). MCP-1-heparin binding was assessed with a biosensor and ELISA. RESULTS AND CONCLUSIONS: Ingramon treatment was accompanied by less pronounced elevation of hsCRP and fibrinogen levels and decreased MCP-1 concentration in plasma in patients after coronary stenting. Ingramon had no effect on MCP-1 interaction with cell receptors or MCP-1 dimerization, but inhibited MCP-1 binding to heparin. The anti-inflammatory activity of the peptide may be mediated by an impaired chemokine interaction with glycosaminoglycans.
Asunto(s)
Angina de Pecho/patología , Quimiocina CCL2/metabolismo , Heparina/metabolismo , Stents , Reacción de Fase Aguda , Anciano , Angioplastia , Antiinflamatorios/farmacología , Proteína C-Reactiva/metabolismo , Angiografía Coronaria/métodos , Reestenosis Coronaria , Femenino , Fibrinógeno/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Monocitos/citología , Isquemia Miocárdica/patología , Fragmentos de Péptidos/farmacología , Unión Proteica , Estructura Terciaria de ProteínaRESUMEN
A multiplex label-free biosensor is developed for diagnostics of autoimmune diseases by highly sensitive measuring in human serum both critical characteristics of autoantibody: concentration and native kinetic parameters that reflect autoantibody aggressiveness to the organism's tissues. The biosensor is based on the spectral-correlation interferometry and image processing of a microarray glass biochip, affordable to be single-used in medical applications. Simultaneous 25-min detection and activity characterization of several autoantibodies in the same serum sample have been demonstrated for anti-thyroglobulin (anti-TG) and anti-thyroid peroxidase (anti-TPO) as models. The biosensor offers extremely high sensitivity: limits of detection in serum are 1.7 IU/mL and 6 IU/mL for anti-TPO and anti-TG, respectively. The dynamic range covers the whole range of clinically relevant concentrations of the autoantibodies up to 1000 IU/mL. The developed method of characterization of autoantibody activity by recording the kinetics of their binding with free native antigens is based on autoantibody polyvalency. The measurements in clinical serum samples have shown that the native kinetic parameters are independent of concentration. The proposed biosensor and method of native kinetic registration can be used to develop new criteria for comprehensive diagnostics of autoimmune diseases, based not only on traditional measurements of concentration but also on quantitative evaluation of autoantibody aggressiveness. The developed method can be adapted to other label-free sensors such as those based on the surface plasmon resonance, optical waveguides, etc.
Asunto(s)
Autoanticuerpos , Técnicas Biosensibles , Inmunoensayo/métodos , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Enfermedades Autoinmunes/sangre , Enfermedades Autoinmunes/diagnóstico , Enfermedades Autoinmunes/inmunología , Humanos , Análisis por Micromatrices/métodosRESUMEN
The data represent in-depth characterization of a novel method for highly sensitive simultaneous measuring in human serum of both critical parameters of autoantibodies: concentration and native kinetics. The latter refers to autoantibody interaction with free, not immobilized, antigen. The method and related biosensors are based on the spectral-correlation and spectral-phase interferometry. The data cover: multi-factor optimization and quantitative characterization of the developed affordable single-used biochips, including X-ray photoelectron spectroscopy (XPS) control of chemical modifications of the surface during fabrication; antibody screening; optimization and verification of protocols for label-free biosensing in human serum; mathematical model for fitting experimental data and calculation of kinetic constants of interaction of autoantibodies with free antigen; comprehensive verification of the method specificity; correlation between the data obtained with the developed biosensor and with enzyme linked immunosorbent assay (ELISA); comparison of analytical characteristics of the developed biosensor with the most advanced label-based methods. The data importance is confirmed by a companion paper (DOI 10.1016/j.bios.2020.112187), which shows that the combination of mentioned autoantibody parameters is promising for more accurate criteria for early diagnostics and efficient therapy of autoimmune disorders. The obtained data can be used in development of a wide range of biosensors, both label-free and based on various labels.
RESUMEN
The effect of triterpene glycosides from holothurians on Na+-K+-ATPase of rat brain was investigated. The marine glycosides are irreversible inhibitors of the enzyme with an average I50 value of 10(-4) M. ATP had a low protective effect against inhibition. The inhibitory effect was increased by preincubation with MgCl2. There was alteration of the activation curve of Na+-K+-ATPase by NaCl and KCl in the presence of glycosides. Triterpene glycosides inhibited the K+-phosphatase activity, but to a smaller degree than the ATPase activity. Na+-K+-ATPase of pig kidney was less sensitive to the marine triterpene glycosides than the brain enzyme. The marine glycosides did not alter the specific binding of [3H]-ouabain to the Na+-K+-ATPase.
Asunto(s)
Encéfalo/enzimología , Glicósidos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Triterpenos/farmacología , Adenosina Trifosfato/farmacología , Animales , Magnesio/farmacología , Cloruro de Magnesio , Ouabaína/farmacología , Potasio/farmacología , Ratas , Pepinos de Mar , Sodio/farmacología , Relación Estructura-ActividadRESUMEN
3,5-Dibromo-1-acetoxy-4-oxo-2,5-cyclohexadien-1-acetonitrile (dienone A) inhibited Na+ - K+-ATPase with a half-maximal inhibition concentration (I50) equal to 2.9 X 10(-6)M. Inhibition was time- and pH-dependent and complete after 20-30 min preincubation within a range of pH from 7.0 to 9.0. Kinetic evaluation of the cationic substrate activation of Na+ - K+-ATPase indicated mixed type inhibition with regard to Na+ and K+ and competitive inhibition with regard to ATP activation of the enzyme. The presence of Mg2+ caused an increased inhibition. Also, K+-p-nitrophenyl phosphatase activity was altered by dienone A and mixed type inhibition with regard to p-nitrophenyl phosphate and K+ was demonstrated. Inhibition was partially restored by repeated washing. Preincubation with sulfhydryl reagents protected the enzyme from inhibition. A significant linear correlation between reactive enzyme sulfhydryl contents [SH] and Na+ - K+-ATPase activity in the presence of varying concentrations of dienone A was observed. One of the factors causing cytotoxic activity of this compound might be its interaction with some thiol groups of the membrane-bound Na+ - K+-ATPase.
Asunto(s)
Acetonitrilos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , 4-Nitrofenilfosfatasa/metabolismo , Adenosina Trifosfato/farmacología , Animales , Ciclohexenos , Activación Enzimática/efectos de los fármacos , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Cinética , Magnesio/farmacología , Poríferos , Potasio/farmacología , Ratas , Sodio/farmacología , Reactivos de Sulfhidrilo/farmacologíaRESUMEN
High-angle X-ray diffraction spectra showed that triterpene glycosides form crystalline complexes with membrane cholesterol. Electron microscopy demonstrated a decreased vesicle size, of the membrane preparation from rat brain which is enriched in Na+-K+-ATPase, by the triterpene glycosides. The Arrhenius plot was linear in the presence of triterpene glycosides. The half-width of the phosphatidylcholine N-methyl proton line in proton NMR spectra was not altered in the presence of marine glycosides. The excimer formation of pyrene, a hydrophobic fluorescent probe, was significantly decreased by triterpene glycosides. The increase of tryptophanyl residue fluorescence demonstrated a change of the Na+-K+-ATPase conformation after treatment with cytotoxic glycosides.
Asunto(s)
Encéfalo/enzimología , Glicósidos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Triterpenos/farmacología , Animales , Encéfalo/ultraestructura , Espectroscopía de Resonancia Magnética , Microscopía Electrónica , Pirenos/análisis , Ratas , Pepinos de Mar , Espectrometría de Fluorescencia , Temperatura , Triptófano/análisis , Difracción de Rayos XRESUMEN
Marine glycosides from the sea cucumbers Actinopyga agassizi, Holothuria atra, Bohadschia argus, Cucumaria fraudatrix, Astichopus multifidus and Thelenota ananas inhibit both Na+-K+ ATPase and Mg2+-ATPase of rat brain in vitro. The glycoside-cholesterol complex of these compounds does not influence ATPase activity. Asterosaponins from starfishes Linckia guildingi and Linckia laevigata possess a slight inhibiting effect. The triterpene glycosides from sea cucumbers are more powerful inhibitors than steroidal glycosides from starfishes.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Glicósidos/farmacología , Toxinas Marinas/farmacología , Animales , Encéfalo/enzimología , Técnicas In Vitro , Microsomas/enzimología , Ratas , Pepinos de Mar , Estrellas de Mar , Relación Estructura-ActividadRESUMEN
The bromine-containing compounds from sponges of the Aplysinidae family inhibit, in vitro, the Na+ -K+ -ATPase activity of the rat brain microsomal fraction. The extent of inhibition is dependent on concentration and chemical structure of the compounds. The substances containing the dienone fragment, such as 3,5-dibromo-1-hydroxy-4-oxo-2,5-cyclohexadien-1-acetamide (IV), 3,5-dibromo-1-acetoxy-4-oxo-2,5-cyclohexadien-1-acetonitrile (V) and 3,5-dibromo-1-hydroxy-4-oxo-2,5-cyclohexadien-1-ethylacetate (VI), are powerful inhibitors of Na+ -K+ -ATPase.
Asunto(s)
Bromo/farmacología , Poríferos/análisis , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Adenosina Trifosfatasas/análisis , Animales , Encéfalo/enzimología , ATPasa de Ca(2+) y Mg(2+) , RatasRESUMEN
Sapogenins from the starfish Asterias amurensis and Lethasterias nanimensis chelifera, 5 alpha-pregn-9(11)-ene-3 beta,6 alpha-diol-20-one, 5 alpha-cholest-9(11)-ene-3 beta,6 alpha-diol-23-one, 5 alpha-cholesta-9(11),24(25)-diene-3 beta,6 alpha-diol-23-one, (20E)-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one and 24 zeta-methyl-5 alpha-cholesta-9(11),20(22)-diene-3 beta,6 alpha-diol-23-one, stimulated the contractile force of the heart of the mollusk Spisula sachalinensis at concentration of 5 x 10(-5) M. Ouabain, a specific inhibitor of Na+,K(+)-ATPase, at concentration of 5 x 10(-5) M had no effect on this physiological model. Starfish sapogenins of the cholestane series moderately inhibited rat brain cortex Na+,K(+)-ATPase and decreased Ca2+ influx into Ehrlich carcinoma cells. In contrast, pregnane asterogenin asterone did not inhibit Na+,K(+)-ATPase and increased the influx of Ca2+ into cells. These effects were not the result of cell membrane damage, because none of the compounds tested have hemolytic activity.
Asunto(s)
Calcio/metabolismo , Corazón/efectos de los fármacos , Sapogeninas/farmacología , Saponinas/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Animales , Encéfalo/efectos de los fármacos , Encéfalo/enzimología , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Colestenonas/farmacología , Inhibidores Enzimáticos/farmacología , Hemólisis/efectos de los fármacos , Humanos , Moluscos , Contracción Miocárdica/efectos de los fármacos , Ouabaína/farmacología , Pregnenos/farmacología , Ratas , Sapogeninas/química , Sapogeninas/aislamiento & purificación , Estrellas de Mar , Esteroles/farmacología , Células Tumorales CultivadasRESUMEN
Effects of two triterpene glycosides, isolated from the holothurian Psolus fabricii, on rat brain Na+, K(+)-ATPase (Na, K-pump; EC 3.6.1.3) were investigated. Psolusosides A and B (PsA and PsB) inhibited rat brain Na+, K(+)-ATPase with I50 values of 1 x 10(-4) M and 3 x 10(-4) M, respectively. PsA significantly stimulated [3H]ATP binding to Na+, K(+)-ATPase, weakly increased [3H]ouabain binding to the enzyme, and inhibited K(+)-phosphatase activity to a smaller degree than the total reaction of ATP hydrolysis. In contrast, PsB decreased [3H]ATP binding to Na+, K(+)-ATPase, and had no effect on [3H]ouabain binding to the enzyme. K(+)-Phosphatase activity was inhibited by PsB in parallel with Na+, K(+)-ATPase activity. The fluorescence intensity of tryptophanyl residues of Na+, K(+)-ATPase was increased by PsA and decreased by PsB in a dose-dependent manner. The excimer formation of pyrene, a hydrophobic fluorescent probe, was decreased by PsA only. The different characteristics of inhibition mode for these substances were explained by peculiarities of their chemical structures and distinctive affinity to membrane cholesterol.
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Corteza Cerebral/enzimología , Equinodermos/química , Glucósidos/farmacología , Glicósidos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Triterpenos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Membrana Celular/enzimología , Inhibidores Enzimáticos , Eritrocitos/metabolismo , Fluorescencia , Humanos , Ouabaína/metabolismo , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Potasio/metabolismo , Conformación Proteica , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Fluorescencia , Triptófano/químicaRESUMEN
Halenaquinol, a natural cardioactive pentacyclic hydroquinone from the sponge Petrosia seriata, was found to be a powerful inhibitor of the rat brainstem and of the rat brain cortex Na+, K(+)-ATPases and the rabbit muscle sarcoplasmic reticulum Ca(2+)-ATPase with I50 values of 7.0 x 10(-7), 1.3 x 10(-6) and 2.5 x 10(-6) M, respectively. Halenaquinol also inhibited K(+)-phosphatase activity of the rat brain cortex Na+, K(+)-ATPase with an I50 value of 3 x 10(-6) M. Ouabain-insensitive Mg(2+)-ATPase activity of the microsomal fraction of the rat brain cortex was weakly inhibited by halenaquinol. Inhibition was irreversible, dose- and time-dependent. Naphthohydroquinone fragment in structures of halenaquinol, related natural and model compounds was very important for an inhibiting effect.
Asunto(s)
Adenosina Trifosfatasas/antagonistas & inhibidores , Benzo(a)Antracenos/química , Benzo(a)Antracenos/farmacología , Inhibidores Enzimáticos/farmacología , Poríferos/química , Animales , Tronco Encefálico/enzimología , ATPasa de Ca(2+) y Mg(2+)/antagonistas & inhibidores , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Membrana Celular/enzimología , Corteza Cerebral/enzimología , Humanos , Contracción Miocárdica/efectos de los fármacos , Monoéster Fosfórico Hidrolasas/antagonistas & inhibidores , Conejos , Rana ridibunda , Ratas , Retículo Sarcoplasmático/enzimología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Relación Estructura-ActividadRESUMEN
A series of 23-oxosteroid derivatives have been synthesized and tested for their inhibiting Na+, K(+)-dependent ATPase from rat brain in the 1 x 10(-6)-1 x 10(-4) M concentrations. Natural 23-oxogenins from sea star Asterias amurensis and synthetic monoesters showed the inhibiting activity upto 50-55%. These compounds caused heart contraction in frogs at the level of the known cardiotonic strophanthin G, and inotropic activity on isolated heart of mollusk Spisula sachalinensis.
Asunto(s)
Glicósidos Cardíacos/farmacología , Contracción Miocárdica/efectos de los fármacos , Animales , Anuros , Encéfalo/efectos de los fármacos , Glicósidos Cardíacos/síntesis química , Técnicas In Vitro , Moluscos , Ratas , Anémonas de Mar , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidoresRESUMEN
A method for effective development of solid-phase immunoassays on a glass surface and for optimization of related protocols by highly sensitive quantitative monitoring of each assay step has been proposed and experimentally implemented. The method is based on the spectral correlation interferometry (SCI) that allows real-time measuring of the thickness of a biomolecular layer bound to the recognition molecular receptors on the sensor chip surface. The method is realized with compact 3-channel SCI-biosensors that employ as the sensor chips standard cover glass slips without deposition of any additional films. Different schemes for antibody immobilization on a glass surface have been experimentally compared and optimized toward a higher sorption capacity of the sensor chips. Comparative characterization of the kinetics of each immunoassay stage has been implemented with the optimized protocols: i) covalent immobilization of antibody on an epoxylated surface and ii) biotinylated antibody sorption on a biotinylated surface via a high-affinity biotin-streptavidin bond. We have shown that magnetic nanoparticles employed as labels with model detection of cardiac troponin I further amplify the SCI signal, resulting in 100-fold improvement of the detection limit. The developed protocols can also be used with the alternative immunoassay platforms, including the label methods based on registration of only the final assay result, which is the quantity of bound labels.
Asunto(s)
Quimiocina CCL2/química , Quimiocina CCL2/metabolismo , Heparina/metabolismo , Técnicas Biosensibles , Interacciones Farmacológicas , Humanos , Técnicas In Vitro , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
Halenaquinol inhibited the partial reactions of ATP hydrolysis by rat brain cortex Na(+),K(+)-ATPase, such as [3H]ATP binding to the enzyme, Na(+)-dependent front-door phosphorylation from [gamma-(33)P]ATP, and also Na(+)- and K(+)-dependent E(1)<-->E(2) conformational transitions of the enzyme. Halenaquinol abolished the positive cooperativity between the Na(+)- and K(+)-binding sites on the enzyme. ATP and sulfhydryl-containing reagents (cysteine and dithiothreitol) protected the Na(+),K(+)-ATPase against inhibition. Halenaquinol can react with additional vital groups in the enzyme after blockage of certain sulfhydryl groups with 5,5'-dithio-bis-nitrobenzoic acid. Halenaquinol inhibited [3H]ouabain binding to Na(+),K(+)-ATPase under phosphorylating and non-phosphorylating conditions. Binding of fluorescein 5'-isothiocyanate to Na(+),K(+)-ATPase and intensity of fluorescence of enzyme tryptophanyl residues were decreased by halenaquinol. We suggest that interaction of halenaquinol with the essential sulfhydryls in/or near the ATP-binding site of Na(+),K(+)-ATPase resulted in a change of protein conformation and subsequent alteration of overall and partial enzymatic reactions.