Presence Of Monovalent Cations Research Articles

Quartets in G‐major

The First International Meeting on Quadruplex DNA took place between 21 and 24 April 2007, in Louisville, Kentucky, USA. The meeting followed two regional workshops (pan‐Pacific and European) in Hawai (2005) and Slovenia (2006), and was organized by J. Chaires and L. Hurley. ![][1] The conference literally started with a bang! The meeting brought together around 110 scientists from all over the world to present their most recent data on quadruplex DNA, and the opening reception coincided with Thunder Over Louisville , a massive fireworks display marking the start of the Kentucky Derby Festival. The focus of the conference was the family of nucleic acid secondary structures known as guanine (G)‐quadruplexes. These can be formed by certain guanine‐rich sequences in the presence of monovalent cations and are stabilized by G‐quartets (Fig 1). Interest in this area has intensified in recent years because G‐quadruplex structures have roles in crucial biological processes and can be targeted for therapeutic intervention (Maizels, 2006; Neidle & Balasubramanian, 2006; Riou, 2004). G‐quadruplexes might also have applications in areas ranging from supramolecular chemistry and nanotechnology to medicinal chemistry (Alberti et al , 2006; Davis, 2004; Petraccone et al , 2005). The aim of this meeting was to assess the current status of this unusual DNA structure, which was discovered more than 40 years ago by our plenary lecturer, D. Davies (Bethesda, MD, USA), and his colleagues (Gellert et al , 1962). The meeting also coincided with the tenth anniversary of a seminal paper that catalysed much interest in the field and laid the foundations for therapeutic possibilities (Sun et al , 1997). Here, we summarize a few of the highlights from the broad range of topics discussed. Figure 1. The quadruplex. (A) A tetrameric, dimeric and monomeric G‐quadruplex composed of three G‐quartets (top). (B) A schematic model of DNA secondary … [1]: /embed/graphic-1.gif

Quadruplex ligands may act as molecular chaperones for tetramolecular quadruplex formation.

G-quadruplexes are a family of four-stranded DNA structures, stabilized by G-quartets, that form in the presence of monovalent cations. Efforts are currently being made to identify ligands that selectively bind to G-quadruplex motifs as these compounds may interfere with the telomere structure, telomere elongation/replication and proliferation of cancer cells. The kinetics of quadruplex–ligands interactions are poorly understood: it is not clear whether quadruplex ligands lock into the preformed structure (i.e. increase the lifetime of the structure by lowering the dissociation constant, koff) or whether ligands actively promote the formation of the complex and act as quadruplex chaperones by increasing the association constant, kon. We studied the effect of a selective quadruplex ligand, a bisquinolinium pyridine dicarboxamide compound called 360A, to distinguish these two possibilities. We demonstrated that, in addition to binding to and locking into preformed quadruplexes, this molecule acted as a chaperone for tetramolecular complexes by acting on kon. This observation has implications for in vitro and in vivo applications of quadruplexes and should be taken into account when evaluating the cellular responses to these agents.

Are liquid crystalline properties of nucleosomes involved in chromosome structure and dynamics?

Nucleosome core particles correspond to the structural units of eukaryotic chromatin. They are charged colloids, 101 Angstrom in diameter and 55 Angstrom in length, formed by the coiling of a 146/147 bp DNA fragment (50 nm) around the histone protein octamer. Solutions of these particles can be concentrated, under osmotic pressure, up to the concentrations found in the nuclei of living cells. In the presence of monovalent cations (Na(+)), nucleosomes self-assemble into crystalline or liquid crystalline phases. A lamello-columnar phase is observed at 'low salt' concentrations, while a two-dimensional hexagonal phase and a three-dimensional quasi-hexagonal phase form at 'high salt' concentrations. We followed the formation of these phases from the dilute isotropic solutions to the ordered phases by combining cryoelectron microscopy and X-ray diffraction analyses. The phase diagram is presented as a function of the monovalent salt concentration and applied osmotic pressure. An alternative method to condense nucleosomes is to induce their aggregation upon addition of divalent or multivalent cations (Mg(2+), spermidine(3+) and spermine(4+)). Ordered phases are also found in the aggregates. We also discuss whether these condensed phases of nucleosomes may be relevant from a biological point of view.

Ribokinase from E. coli: Expression, purification, and substrate specificity

Ribokinase (RK) was expressed in the Escherichia coli ER2566 cells harboring the constructed expression plasmid encompassing the rbsK gene, encoding ribokinase. The recombinant enzyme was purified from sonicated cells by double chromatography to afford a preparation that was ca. 90% pure and had specific activity of 75 μmol/min mg protein. Catalytic activity of RK: (i) is strongly dependent on the presence of monovalent cations (potassium ⋙ ammonium > cesium), and (ii) is cooperatively enhanced by divalent magnesium and manganese ions. Besides d-ribose and 2-deoxy- d-ribose, RK was found to catalyze the 5- O-phosphorylation of d-arabinose, d-xylose, and d-fructose in the presence of ATP, and potassium and magnesium ions; l-ribose and l-arabinose are not substrates for the recombinant enzyme. A new radiochemical method for monitoring the formation of d-pentofuranose-5-[ 32P]phosphates in the presence of [γ- 32P]ATP and RK is reported.

Structural switch of telomere DNA by pH and monovalent cation

Because of the importance of telomere DNAs, the structures of these DNAs in vivo and in vitro are currently of great research interest in the medical, pharmaceutical, chemical, and industrial fields. In this study, we investigated the structure and thermodynamic properties of the telomere DNAs in the presence of monovalent cations, K+ or Na+. The results demonstrate that the duplex-quadruplex conversion of the 1:1 mixture of telomere G-rich and C-rich sequences can be induced depending on cation species and its concentration. Furthermore, slightly acidic condition stabilizes the i-motif and forces the mixture to dissociate. These results suggest that the structure and stability of telomere DNAs can be controlled by monovalent cation and pH.

Locating Counterions in Guanosine Quadruplexes: A Contrast-Variation Neutron Diffraction Experiment in Condensed Hexagonal Phase

Guanosine, one of the primary components of nucleic acids, self-associates in water to form G-quadruplexes, four-stranded helicoidal aggregates, made by stacked planar tetramers, consisting of four planar guanine molecules. Essential for the stability of these supramolecular aggregates is the presence of monovalent cations. As G-quadruplexes show a lyotropic polymorphism, neutron diffraction, in combination with the H2O/D2O contrast variation technique, has been applied to study the cation structural features of quadruplexes in hexagonal phase at different hydrations and counterion concentrations. The guanosine 5'-monophosphate, dipotassium salt, was considered and G-quadruplexes in hexagonal phase were prepared in the different experimental conditions (contrast, hydration and KCl solution concentration) by using the osmotic stress technique. The calculated scattering length density distribution maps show that counterions fill the helix inner cavity and that atmospheric cations are bound to a second site, close to the external phosphate groups. The site occupancy turned out to be very high: we found on the inner site 0.87 K ions per tetramer in G-quadruplexes prepared in pure water and 0.97 K ions per tetramer in G-quadruplexes prepared in KCl 0.5 M, while in all cases 6 ions per unit cell were detected to occupy the outer sites, partially neutralizing the two formal negative charges per phosphate group. The very large K ions concentration difference between the binding sites and the bulk solution demonstrates that counterions are structurally involved in the formation and in the stabilization of the helices.

The reaction of indole with the aminoacrylate intermediate of Salmonella typhimurium tryptophan synthase: observation of a primary kinetic isotope effect with 3-[ 2H]indole

The bacterial tryptophan synthase α 2β 2 complex catalyzes the final reactions in the biosynthesis of l-tryptophan. Indole is produced at the active site of the α-subunit and is transferred through a 25–30 Å tunnel to the β-active site, where it reacts with an aminoacrylate intermediate. Lane and Kirschner [Eur. J. Biochem. 129 (1983) 571] proposed a two-step nucleophilic addition–tautomerization mechanism for the reaction of indole with the aminoacrylate intermediate, based on the absence of an observed kinetic isotope effect (KIE) when 3-[ 2H]indole reacts with the aminoacrylate intermediate. We have now observed a KIE of 1.4–2.0 in the reaction of 3-[ 2H]indole with the aminoacrylate intermediate in the presence of monovalent cations, but not when an α-subunit ligand, disodium α-glycerophosphate (Na 2GP), is present. Rapid-scanning stopped flow kinetic studies were performed of the reaction of indole and 3-[ 2H]indole with tryptophan synthase preincubated with l-serine, following the decay of the aminoacrylate intermediate at 350 nm, the formation of the quinonoid intermediate at 476 nm, and the formation of the l-Trp external aldimine at 423 nm. The addition of Na 2GP dramatically slows the rate of reaction of indole with the α-aminoacrylate intermediate. A primary KIE is not observed in the reaction of 3-[ 2H]indole with the aminoacrylate complex of tryptophan synthase in the presence of Na 2GP, suggesting binding of indole with tryptophan synthase is rate limiting under these conditions. The reaction of 2-methylindole does not show a KIE, either in the presence of Na + or Na 2GP. These results support the previously proposed mechanism for the β-reaction of tryptophan synthase, but suggest that the rate limiting step in quinonoid intermediate formation from indole and the aminoacrylate intermediate is deprotonation.

Detection of quadruplex DNA structures in human telomeres by a fluorescent carbazole derivative.

Single-stranded telomeric DNA tends to form a four-base-paired planar structure termed G-quadruplex. This structure was easily formed in vitro in the presence of monovalent cations. However, the existence of this structure in native human telomeres is unclear. Here we address this important question through the distinctive properties of 3,6-bis(1-methyl-4-vinylpyridinium)carbazole diiodide (BMVC) upon binding to various DNA structures. Although the fluorescence of BMVC increases significantly in the presence of DNA, BMVC has high sensitivity and binding preference to quadruplex d(T(2)AG(3))(4) over duplex DNA. In addition, the fluorescent emissions were characterized around 575 nm for quadruplex d(T(2)AG(3))(4) and 545 nm for most of duplex DNA. The 575-nm fluorescence emissions were detected in the mixtures of 2 nM BMVC with the chromosomal DNA that were extracted from human cells, suggesting the presence of quadruplex structure in human nucleus. Further analyzing the BMVC fluorescence at the ends of metaphase chromosomes and other regions of chromosomes, we detected the quadruplex-binding BMVC fluorescence at telomere-proximal regions. Together these results provide the first evidence for the presence of quadruplex structures in human telomeres.

Self-association of cromolyn sodium in aqueous solution characterized by nuclear magnetic resonance spectroscopy

The major objective of this study was to investigate and characterize the solution properties of cromolyn sodium (in D2O or D2O/H2O phosphate buffer at pH 7.5) using nuclear magnetic resonance (NMR) spectroscopy. The self-association of cromolyn molecules was examined primarily via one-dimensional 1H and 13C, and two-dimensional homonuclear NOESY NMR. Significant spectral shifts were observed for a majority of cromolyn 1H and 13C resonances, and are attributed to inter-molecular ring-stacking association accompanied by intra-molecular conformational changes. The critical self-association concentration was determined to be 10 mg/mL at pH 7.5 and 25°C by measuring the chemical shift of a specific cromolyn 1H resonance. The observed magnitude and sign changes of NOESY correlations indicate the formation of cromolyn aggregates with restricted molecular mobility. Mesomorphic liquid crystal formation is suggested by uniformly pronounced line broadening in concentrated cromolyn solutions; the transition concentration was approximately 60 mg/mL at 25°C, which is consistent with literature findings based on other techniques. A stronger tendency toward association was observed at lower temperature but aggregation appeared to be independent of pH. Lastly, it was concluded that self-association of cromolyn is promoted by the presence of monovalent cations as a result of reduced electrostatic repulsive forces. © 2004 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 93:1351–1358, 2004

Direct hydrothermal synthesis of metal intercalated hexagonal molybdates, M + x Mo6−x/3O18−x (OH)x.yH2O (M = Li, Rb, Cs, NH4)

Here we report direct hydrothermal synthesis of a few hexagonal molybdates with composition, M x /+ Mo 6−x Emphasis>/3/6+ O18−x (OH) x .yH2O (M = Li, Rb, Cs, NH4). The molybdates crystallize in the space groupP6 3 /m with a r∼ 10.5 andc r∼ 3.7 A . Unlike previous studies, our work suggests that hexagonal molybdates could be stabilized in the presence of monovalent cations with varying ionic size (smaller lithium to larger cesium) under hydrothermal condition. The phases showed exceptional thermal stability till 550°C.

Evidence for substrate activation of electron transfer from methylamine dehydrogenase to amicyanin.

Electron transfer (ET) from methylamine dehydrogenase (MADH) to amicyanin may be true or gated ET, depending upon the redox form of MADH. ET from the substrate-reduced aminoquinol form of MADH is gated, and the reaction rate is dependent on the presence of monovalent cations. This ET reaction has been studied in buffer free of monovalent cations. The reaction rate is orders of magnitude less than with saturating concentrations of monovalent cation. Analysis of the temperature dependence of this slow reaction, however, reveals that it is a true ET reaction. The rate of MADH reduction by substrate and the steady-state rate of substrate-dependent reduction of amicyanin by MADH were examined in different buffers. The results reveal that, in the steady state, the protonated methylammonium substrate performs the role previously attributed to monovalent cations in regulating the rate and mechanism of ET from MADH. The two putative cation binding sites previously observed in the crystal structure of MADH may now be assigned distinct roles, one as a catalytic substrate binding site and the other as a noncatalytic regulatory substrate binding site.

Model Protein BSA Adsorption and Covalent Coupling onto Methyl Methacrylate Based Latex Particles with Different Surface Properties

The adsorption and covalent coupling of bovine serum albumin (BSA) onto methylmethacrylate (MMA) based monodisperse latex particles with different hydrophylic surfaces were investigated. P(MMA) and P(MMA/HEMA) microbeads in the size range of 1.5–2.0 mm were prepared by a dispersion homopolymerization method in the presence of poly(vinylpyrrolidone) (PVP) and poly(vinyl alcohol) (PVA) as steric stabilizer and by copolymerization of MMA and 2-hydroxethyl methacrylate (HEMA) where PVP was used as steric stabilizers. Surface properties of these particles were characterized in terms of contact angle measurements and FTIR-DRS spectra. Additionally, hydroxyl groups of PMMAPVA and P(MMA/HEMA) particles were activated to provide aldehyde groups on surfaces. Adsorption of the BSA onto these five types of latex particles was examined as a function of initial albumin concentration, pH and ionic strength of the various cations (Na+,Ca+ and Mg− and anions (Cl− and SCN−). The maximum adsorption capacities for all latex particles were obtained in solutions with 0.01μ m ionic strength and 1 mg/mL BSA concentration. When using PMMAPVA, P(MMA/HEMA) and PMMAPVP, the adsorption increased as hydrophobicity increased. Aldehyde activation of the two most hydrophobic particles elevated the coupling (immobilization) significantly in ionic strength of 0.01μ m. The adsorption and/or immobilization were maximum in the presence of monovalent cation, Na+ or anion, Cl−B for all types of latex particles.

Kinetics of the Protonation of Macrocyclic Amines in the Presence of Monovalent Cations in Aqueous Solution

For macrocyclic bases such as: 1-aza-15-crown-5 (N15C5),1,4,10-trioxa-7,13-diazacyclopentadecan (21), 1,7,10,16-tetraoxa-4,13-diazacyclooctadecan (22) and 1, 4, 7, 13, 16-pentaoxa-10, 19-diazacycloheneicosane (23), the kinetics of deprotonation and protonation reactions in the presence of monovalent cations was studied using the temperature jump technique. For the sake of comparison, the measurements were also performed for 1,4-diazabicyclo[2,2,2]octane (DABCO) base, which does not form complexes with monovalent cations. The monovalent cations affect the temperature dependence of the kinetic parameters of deprotonation. They also affect the activation parameters, which is shown by a distribution of ΔH≠ and ΔS≠ values, but do not influence the value of ΔG≠.

Conformational behavior of nucleotide-sugar in solution: molecular dynamics and NMR study of solvated uridine diphosphate-glucose in the presence of monovalent cations.

The nucleotide-sugars are metabolites of primary importance in the biosynthesis of polysaccharides and glycoconjugates since they serve as sugar donors in the reactions of glycosyltransferases, enzymes that displays a high specificity for both donors and acceptors. In order to determine the conformational behavior of uridinediphosphoglucose in dilute aqueous solution that includes a physiologically relevant concentration of salt, parallel NMR and molecular modeling investigations have been conducted. Nine molecular dynamics trajectories of 3 ns each were calculated in presence of explicit water and monovalent cations with the use of the AMBER force field with recently developed energy parameters for nucleotide-sugars (P. Petrova, J. Koca, and A. Imberty, Journal of American Chemical Society, 1999, vol. 121, pp. 5535-5547). Theoretical nuclear Overhauser effect data were calculated from these simulations using a model-free approach that takes into account internal motions. Comparison of theoretical and experimental data gives excellent agreement for the region surrounding the glucose-phosphate linkage including the pyrophosphate linkage itself. Less satisfactory agreement is obtained for the ribose ring and the base orientations. On the whole, both NMR and molecular dynamics simulations predict the molecule to be flexible, and to visit a large number of conformations while maintaining an extended overall shape.

Mechanisms of monovalent cation action in enzyme catalysis: the first stage of the tryptophan synthase beta-reaction.

The tryptophan synthase bienzyme complex is activated and regulated by the allosteric action of monovalent cations (MVCs). The kinetic dissection of the first stage (stage I) in the beta-reaction of tryptophan synthase, the reaction of L-serine with pyridoxal 5'-phosphate at the beta-site to give the alpha-aminoacrylate Schiff base intermediate, E(A-A), is here examined in the absence and presence of MVCs. This analysis reveals which of the individual steps are greatly affected in stage I and how the ground states and transition states are affected by MVCs. Kinetic studies in combination with a detailed relaxation kinetic analysis to determine the specific rate constants for the conversion of the L-Ser external aldimine, E(Aex1), to E(A-A) show that the primary kinetic isotope effect for proton abstraction from Calpha of the E(Aex1) species changes from 4.0 +/- 0.4 in the absence of MVCs to a value of 5.9 +/- 0.5 in the presence of Na+, indicating that the nature of the transition state for this C-H scission is significantly perturbed by the MVC effect. The E(A-A) species was found to exist in two conformations with different activities, the ratio of which is affected by the presence of MVCs. It is shown that changes in the rate constants of stage I are important in establishing the ratio of active to inactive conformations of the E(A-A) species. Consequently, the MVC effect alters the relative energies of both the transition states and the ground states for selected steps in stage I of the pathway. Hence, interactions at the MVC site give rise to a fine-tuning of the covalent bonding interactions between active site residues and the reacting substrate during the conformational cycle of the bienzyme complex.

Mechanisms of monovalent cation action in enzyme catalysis: the tryptophan synthase alpha-, beta-, and alpha beta-reactions.

The alpha-subunit of the tryptophan synthase bienzyme complex catalyzes the formation of indole from the cleavage of 3-indolyl-D-glyceraldehyde 3'-phosphate, while the beta-subunit utilizes L-serine and the indole produced at the alpha-site to form tryptophan. The replacement reaction catalyzed by the beta-subunit requires pyridoxal 5'-phosphate (PLP) as a cofactor. The beta-reaction occurs in two stages: in stage I, the first substrate, L-Ser, reacts with the enzyme-bound PLP cofactor to form an equilibrating mixture of the L-Ser Schiff base, E(Aex1), and the alpha-aminoacrylate Schiff base intermediate, E(A-A); in stage II, this intermediate reacts with the second substrate, indole, to form tryptophan. Monovalent cations (MVCs) are effectors of these processes [Woehl, E., and Dunn, M. F. (1995) Biochemistry 34, 9466-9476]. Herein, detailed kinetic dissections of stage II are described in the absence and in the presence of MVCs. The analyses presented complement the results of the preceding paper [Woehl, E., and Dunn, M. F. (1999) Biochemistry 38, XXXX-XXXX], which examines stage I, and confirm that the chemical and conformational processes in stage I establish the presence of two slowly interconverting conformations of E(A-A) that exhibit different reactivities in stage II. The pattern of kinetic isotope effects on the overall activity of the beta-reaction shows an MVC-mediated change in rate-limiting steps. In the absence of MVCs, the reaction of E(A-A) with indole becomes the rate-limiting step. In the presence of Na+ or K+, the conversion of E(Aex1) to E(A-A) is rate limiting, whereas some third process not subject to an isotope effect becomes rate determining for the NH4+-activated enzyme. The combined results from the preceding paper and from this study define the MVC effects, both for the reaction catalyzed by the beta-subunit and for the allosteric communication between the alpha- and beta-sites. Partial reaction-coordinate free energy diagrams and simulation studies of MVC effects on the proposed mechanism of the beta-reaction are presented.

On the use of hexacyanocobaltates in the separation of caesium from radioactive waste solutions

This paper reviews our work on the use of hexacyanocobaltate salts in the chemical treatment of radioactive waste solutions. The divalent cations of the 3d-block elements together with the hexacyanocobaltate moiety form insoluble polymeric coordination compounds. These compounds have been used as chemical precipitants or ion exchangers for the separation of radiocesium from various simulated and actual waste solutions. Precipitation in situ has been performed by using various hexacyanocobaltate salts of the divalent cations of Mn, Fe, Co and Ni. The results showed that Co2+ and Ni2+ salts have high selectivity for caesium. The presence of monovalent cations and detergent has significant effect on the precipitation process. On the other hand Cd2+ salt has been found to be more stable than other salts in alkaline medium. As ion exchanger the sorption properties of hexacyanocobaltate salts were studied. Adsorption of a cesium seems to proceed via an ion exchange mechanism. It has been used for the treatment of reprocessing medium active waste solution. A decontamination factor of about 103 has been achieved.

Telomeric interactions result in the formation of intramolecular circles behaving as topologically constrained

The gene-sized macronuclear DNA molecules of hypotrichous ciliates carry telomeric sequences of homogenous length. Incubation of these molecules at low concentrations in the presence of monovalent cations (K +, Na +, Cs +) leads to the formation of intramolecular circles. They can be visualized on one or two-dimensional agarose gels only when KCl is present in the gel. From their electrophoretic behavior on agarose gels as well as on density gradients it can be concluded that they are topologically constrained. Digestion of macronuclear DNA with S 1as well as various exonucleases indicate that both the 3′ overhang and the 5′ C-rich strand of the telomeric repeat is involved in these interactions. Several models of these interactions are discussed.

The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Photolabile Amiloride Derivatives as Cation Site Probes of the Na,K-ATPase

Treatment of purified canine renal Na,K-ATPase with a range of photoactivatable amiloride derivatives results in inhibition of ATPase activity prior to illumination. Inhibition by amiloride derivatives substituted on a guanidium N could not be prevented by the presence of either K or Na; however, these cations could protect the enzyme against inhibition by derivatives substituted on the 5-position of the pyrazine ring. In the case of 5-(N-ethyl-[2'-methoxy-4'-nitrobenzyl])amiloride (NENMBA), the presence of monovalent cations (Na, K, and Rb) protected the enzyme effectively against inhibition, with concentrations in the millimolar range. ATP did not prevent inhibition; furthermore, native and NENMBA-treated enzyme exhibited normal levels of high affinity [3H]ADP (and hence ATP) binding. The rate of inhibition increased with increasing concentrations of NENMBA. Extensive washing of NENMBA-inhibited enzyme did not restore ATPase activity, showing that NENMBA has an extremely slow off-rate for dissociation from its inhibitory site. Partially inhibited enzyme could be rapidly pelleted and resuspended in NENMBA-free buffer and inhibition was observed to continue, albeit at a somewhat diminished rate, suggesting that NENMBA gains access to its inhibitory site after partitioning into the lipid phase rather than directly from the aqueous solution. Photolysis of NENMBA-inhibited enzyme resulted in covalent incorporation of the reagent into the alpha-subunit of the Na,K-ATPase, as observed by separation of labeled protein on a Laemmli gel and Western analysis using a polyclonal amiloride antibody. Almost all of the covalent labeling could be prevented by the presence of Rb in the incubation and labeling medium. These results suggest that NENMBA inhibits the Na, K-ATPase by disruption of the cation transport domain rather than the catalytic domain of the enzyme and that it promises to be a useful tool for cation site localization.