Absence Of Na Research Articles

Sodium and water homeostasis is maintained in rats with chronic TRPV4 blockade

The transient receptor potential vanilloid 4 (TRPV4) is a non-selective cation channel that highly expressed in renal tubules. Co-localization of TRPV4 with sodium transporters suggests that TRPV4 might have a role in the regulation of sodium and water homeostasis. To investigate this potential interaction, we determined the blood pressure and renal responses to chronic TRPV4 blockade (GSK2193874A, 30 mg/kg/d) in rats in the presence and absence of Na, K,2Cl− cotransporter, Na, Cl cotransporter, or arginine vasopressin V2 receptor (AVP-V2) inhibition. Under normal conditions, TRPV4 blockade had no effect on mean arterial pressure, renal excretory function, free water clearance, or plasma sodium concentrations. The diuretic and natriuretic responses to Na, K,2Cl− cotransporter inhibition (furosemide, 30 mg/kg) or Na, Cl cotransporter inhibition (hydrochlorothiazide, 30 mg/kg) were unaltered after 5 days of TRPV4 blockade. In contrast, GSK2193874A increased the diuretic, natriuretic, and plasma sodium responses to AVP-V2 inhibition (tolvaptan, 10 mg/kg). Further investigation showed that GSK2193874A increased the plasma concentration of tolvaptan which accounted for the enhanced renal response. In conclusion, TRPV4 has no major effect on blood pressure or sodium and water homeostasis in normal rats either directly or through modulation of sodium transporter or AVP-V2 function in the kidney.

Synthesis of high-silica CHA type zeolite by interzeolite conversion of FAU type zeolite in the presence of seed crystals

The influence of seed crystals on the interzeolite conversion of FAU type zeolite into CHA type zeolite was investigated in the presence of benzyltrimethylammonium hydroxide as a structure-directing agent under various hydrothermal synthesis conditions. Pure and highly crystalline CHA type zeolites with a wide range of Si/Al ratios were obtained in a shorter crystallization time as compared with those obtained without seed crystals. Furthermore, we achieved the first successful synthesis of high-silica CHA type zeolite in the absence of Na + cations by increasing the seed content. The protonated CHA type zeolite with a Si/Al ratio of ca. 15 yielded the highest propylene yield of ca. 48 C-% in ethanol conversion into light olefins.

Loss-of-function mutations in sodium channel Nav1.7 cause anosmia.

Loss of function of the gene SCN9A, encoding the voltage-gated sodium channel Na(v)1.7, causes a congenital inability to experience pain in humans. Here we show that Na(v)1.7 is not only necessary for pain sensation but is also an essential requirement for odour perception in both mice and humans. We examined human patients with loss-of-function mutations in SCN9A and show that they are unable to sense odours. To establish the essential role of Na(v)1.7 in odour perception, we generated conditional null mice in which Na(v)1.7 was removed from all olfactory sensory neurons. In the absence of Na(v)1.7, these neurons still produce odour-evoked action potentials but fail to initiate synaptic signalling from their axon terminals at the first synapse in the olfactory system. The mutant mice no longer display vital, odour-guided behaviours such as innate odour recognition and avoidance, short-term odour learning, and maternal pup retrieval. Our study creates a mouse model of congenital general anosmia and provides new strategies to explore the genetic basis of the human sense of smell.

Cytosolic zinc release and clearance in hippocampal neurons exposed to glutamate – the role of pH and sodium

Although Zn(2+) homeostasis in neurons is tightly regulated and its destabilization has been linked to a number of pathologies including Alzheimer's disease and ischemic neuronal death, the primary mechanisms affecting intracellular Zn(2+) concentration ([Zn(2+) ](i)) in neurons exposed to excitotoxic stimuli remain poorly understood. The present work addressed these mechanisms in cultured hippocampal neurons exposed to glutamate and glycine (Glu/Gly). [Zn(2+)](i) and intracellular Ca(2+) concentration were monitored simultaneously using FluoZin-3 and Fura-2FF, and intracellular pH (pH(i)) was studied in parallel experiments using 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein. Glu/Gly applications under Na(+)-free conditions (Na(+) substituted with N-methyl-D-glucamine(+)) caused Ca(2+) influx, pH(i) drop, and Zn(2+) release from intracellular stores. Experimental maneuvers resulting in a pH(i) increase during Glu/Gly applications, such as stimulation of Na(+) -dependent pathways of H(+) efflux, forcing H(+) efflux via gramicidin-formed channels, or increasing extracellular pH counteracted [Zn(2+)](i) elevations. In the absence of Na(+), the rate of [Zn(2+)](i) decrease could be correlated with the rate of pH(i) increase. In the presence of Na(+), the rate of [Zn(2+) ](i) decrease was about twice as fast as expected from the rate of pH(i) elevation. The data suggest that Glu/Gly-induced cytosolic acidification promotes [Zn(2+) ](i) elevations and that Na(+) counteracts the latter by promoting pH(i)-dependent and pH(i)-independent mechanisms of cytosolic Zn(2+) clearance.

Is neuronal communication with NG2 cells synaptic or extrasynaptic?

NG2-expressing glial cells (NG2 cells) represent a major pool of progenitors able to generate myelinating oligodendrocytes, and perhaps astrocytes and neurones, in the postnatal brain. In the last decade, it has been demonstrated that NG2 cells receive functional glutamatergic and GABAergic synapses mediating fast synaptic transmission in different brain regions. However, several controversies exist in this field. While two classes of NG2 cells have been defined by the presence or absence of Na(+) channels, action potential firing and neuronal input, other studies suggest that all NG2 cells possess Na(+) conductances and are the target of quantal neuronal release, but are unable to trigger action potential firing. Here we bring new evidence supporting the idea that the level of expression of Na(+) conductances is not a criterion to discriminate NG2 cell subpopulations in the somatosensory cortex. Surprisingly, recent reports demonstrated that NG2 cells detect quantal glutamate release from unmyelinated axons in white matter regions. Yet, it is difficult from these studies to establish whether axonal vesicular release in white matter occurs at genuine synaptic junctions or at ectopic release sites. In addition, we recently reported a new mode of extrasynaptic communication between neurones and NG2 cells that relies on pure GABA spillover and does not require GABAergic synaptic input. This review discusses the properties of quantal neuronal release onto NG2 cells and gives an extended overview of potential extrasynaptic modes of transmission, from ectopic to diffuse volume transmission, between neurones and NG2 cells in the brain.

Decoupling of the Catalytic and Transport Activities of Complex I from Rhodothermus marinus by Sodium/Proton Antiporter Inhibitor

The energy transduction by complex I from Rhodothermusmarinus was addressed by studying the influence of 5-(N-ethyl-N-isopropyl)-amiloride (EIPA) on the activities of this enzyme. EIPA is an inhibitor of both Na(+)/H(+) antiporter and complex I NADH:quinone oxidoreductase activity. We performed studies of NADH:quinone oxidoreductase and H(+) and Na(+) translocation activities of complex I from R. marinus at different concentrations of EIPA, using inside-out membrane vesicles. We observed that the oxidoreductase activity and both H(+) and Na(+) transports are inhibited by EIPA. Most interestingly, the catalytic and the two transport activities showed different inhibition profiles. The transports are inhibited at concentrations of EIPA at which the catalytic activity is not affected. In this way the catalytic and transport activities were decoupled. Moreover, the inhibition of the catalytic activity was not influenced by the presence of Na(+), whereas the transport of H(+) showed different inhibition behaviors in the presence and absence of Na(+). Taken together our observations indicate that complex I from R. marinus performs energy transduction by two different processes: proton pumping and Na(+)/H(+) antiporting. The decoupling of the catalytic and transport activities suggests the involvement of an indirect coupling mechanism, possibly through conformational changes.

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.

Determination of the Na +/Glucose Cotransporter (SGLT1) Turnover Rate using the Ion-Trap Technique

The Na+/glucose cotransporter (SGLT1) is a membrane protein that couples the transport of 2 Na+ ions and 1 glucose molecule using the “alternating access mechanism”. This mechanism involves the cotransporter oscillating between two conformations characterized by intracellular versus extracellular accessibility to the binding sites. The turnover rate (TOR) is the number of complete cycles that each cotransporter performs per second. It is a crucial parameter to establish since it can be used to estimate the number of cotransporters present and it sets a lower limit for the slowest rate constant involved in the cotransport mechanism (conformational changes, binding and de-binding reactions). In this study, we obtained an independent estimate of the TOR for human SGLT1 expressed in Xenopus laevis oocytes using a new approach: the Ion-Trap technique (ITT, Blanchard MG. et al. AJP 2008;295(5):C1464-72.) where an extracellular ion-selective electrode of large diameter is used to detect the quantity of ions that are rapidly taken up by the cotransporter (within 20 ms) when the binding sites are suddenly exposed to the extracellular solution by application of a negative membrane potential. Taking advantage of the fact that hSGLT1, in the absence of Na+, can cotransport glucose with protons, we used a pH electrode to determine a TOR of 8.00 ± 1.3 s−1 in the presence of 35 mM α-methyl-glucose (αMG) at −150 mV (pH 5.5). This can be used to calculate a TOR of 13.3 ± 2.4 s−1, for the same group of oocytes, under near Vmax conditions i.e. in the presence of 90 mM Na+ and 5 mM αMG. Under these circumstances, the average cotransport current was −1.08 ± 0.61 μA (n=14) and this activity was generated by an average of 3.6 ± 0.7 ×1011 cotransporter molecules per oocyte.

The Cytoplasmic Ion Permeation Pathway of the Cardiac Na +-Ca 2+ Exchanger

The Na+-Ca2+ Exchanger (NCX) is a plasma membrane protein that exchanges three Na+ for one Ca2+. Transport function has been associated with two regions of NCX that have similar sequence: the α-repeats. Mutations here alter the transport properties of NCX. These two regions, which consist of residues spanning TMSs 2 and 3 (α-1 repeat) and a portion of TMS 7 with the following intracellular loop (α-2 repeat), are modeled to be in close proximity and may form an ion conduction pathway. The goal of this work was to understand transport function and define the pathway through which Na+ and Ca2+ diffuse from the cytoplasm to their intracellular binding sites. We mutated TMS 2 residues to cysteine and determined the affinities of these mutants for Na+ and Ca2+. Also, the reactivity of cysteines to MTSET was investigated to demonstrate intracellular accessibility. Of mutations at 10 positions, 3 behaved like the wild-type exchanger (A106C, P112A, S117C). Mutant M105C had altered affinity for Na+ but not Ca2+ and, like A106C, was blocked by cytoplasmic MTSET in the presence or absence of Na+. In contrast, Na+ blocked the sensitivity of L107C, S109C and S110C to MTSET. This suggests that Na+ binding prevents the cytoplasmic accessibility of these residues. Additionally, mutations at L107, S109 and S110 had altered affinities for intracellular Na+ and Ca2+. For S110C, the selectivity properties of NCX were also altered allowing Li+/Ca2+ exchange. We conclude that TMS 2 is involved in ion binding and transport and is accessible to cytoplasmic sulfhydryl agents in a substrate-dependent manner. The data also suggest the presence of an aqueous cavity used by Na+ and Ca2+ to reach their binding sites involving TMS 2.

L-Serine Decreases Taurine Concentration in the Extracellular Fluid of Brain Slices

L-Serine is considered a functional amino acid in the central nervous system, and induces sedation and hypnotic effects in some animal models of acute and chronic stress. Accordingly, while L-serine is a candidate anti-stress factor, the central mechanism of L-serine is not clear. The present study clarifies the action of L-serine using acute chick brain slices. We investigated the changes in some extracellular fluid amino acid concentrations in response to L-serine perfusion. Taurine concentration decreased while L-alanine concentration increased following L-serine perfusion. To examine the involvement of the taurine transporter, the effect of L-serine on the taurine concentration in the presence and absence of Na+ was also investigated. Na+ had no effect on taurine concentration induced by L-serine perfusion. These results suggest that L-serine has an ability to promote L-alanine synthesis facilitating the catabolism of taurine. In conclusion, L-serine modifies the metabolism of taurine and L-alanine in the extracellular space in chick brain.

Determination of the Na+/Glucose Cotransporter (SGLT1) Turnover Rate Using the Ion-Trap Technique

The Na+/glucose cotransporter (SGLT1) is a membrane protein that couples the transport of two Na+ ions and one glucose molecule using the so-called alternating access mechanism. According to this principle, each cotransporter molecule can adopt either of two main conformations: one with the binding sites accessible to the extracellular solution and one with the binding sites facing the intracellular solution. The turnover rate (TOR) is the number of complete cycles that each protein performs per second. Determination of the TOR has important consequences for investigation of the cotransport mechanism, as none of the rate constants involved in mediating transport in a given direction (conformational changes and binding and unbinding reactions) can be slower than the TOR measured under the same conditions. In addition, the TOR can be used to estimate the number of cotransporter molecules involved in generating a given ensemble activity. In this study, we obtain an independent estimation of the TOR for human SGLT1 expressed in Xenopus laevis oocytes applying the ion-trap technique. This approach detects the quantity of ions released in or taken up from the restricted space existing between the oocyte plasma membrane and the tip of a large ion-selective electrode. Taking advantage of the fact that hSGLT1 in the absence of Na+ can cotransport glucose with protons, we used a pH electrode to determine a TOR of 8.00 ± 1.3 s−1 in the presence of 35 mM α-methyl-glucose at −150 mV (pH 5.5). For the same group of oocytes, a TOR of 13.3 ± 2.4 s−1 was estimated under near-Vmax conditions, i.e., in the presence of 90 mM Na+ and 5 mM α-methyl-glucose. Under these circumstances, the average cotransport current was −1.08 ± 0.61 μA (n = 14), and this activity was generated by an average of 3.6 ± 0.7 × 1011 cotransporter molecules/oocyte.

Proton Diet for the Sodium Pump,

In the absence of Na(+) and K(+) ions the Na,K-ATPase shows a pH-dependent ATP hydrolysis that can be inhibited by ouabain. At pH 7.2 this activity is 5% of the maximal under physiological conditions. It could be inferred that this activity is associated with H(+) transport in both directions across the membrane and facilitates an H-only mode of the sodium pump under such unphysiological conditions. By the analysis of experiments with reconstituted proteoliposomes an overall electroneutral transport mode has been proven. The stoichiometry was determined to be 2 H(+)/2 H(+)/1 ATP and is comparable to what is known from the closely related H,K-ATPase. By time-resolved ATP-concentration jump experiments it was found that at no time was the third, Na(+)-specific binding site of the pump occupied by protons. A modified Post-Albers pump cycle is proposed, with H(+) ions as congeners for Na(+) and K(+), by which all experiments performed can be explained.

A Structural Determinant for the Control of PIP2 Sensitivity in G Protein-gated Inward Rectifier K+ Channels

Inward rectifier K+ (Kir) channels are activated by phosphatidylinositol-(4,5)-bisphosphate (PIP2), but G protein-gated Kir (KG) channels further require either G protein βγ subunits (Gβγ) or intracellular Na+ for their activation. To reveal the mechanism(s) underlying this regulation, we compared the crystal structures of the cytoplasmic domain of KG channel subunit Kir3.2 obtained in the presence and the absence of Na+. The Na+-free Kir3.2, but not the Na+-plus Kir3.2, possessed an ionic bond connecting the N terminus and the CD loop of the C terminus. Functional analyses revealed that the ionic bond between His-69 on the N terminus and Asp-228 on the CD loop, which are known to be critically involved in Gβγ- and Na+-dependent activation, lowered PIP2 sensitivity. The conservation of these residues within the KG channel family indicates that the ionic bond is a character that maintains the channels in a closed state by controlling the PIP2 sensitivity.

Effect of functional unloading and the deficit of dystrophin on the local hyperpolarization of the postsynaptic membrane of a skeletal muscle fiber

The data obtained confirm the unity of the electrogenic mechanism of hyperpolarization by nanomolar concentrations of cholinergic ligands in the extrasynaptic region and by endogenous nonquantal acetylcholine in the synaptic region of a skeletal muscle fiber. In both cases, this mechanism involves the α2 isoform of Na,K-ATPase and is operative in the absence of Na+ entry through membrane channels. At the same time, there are peculiar properties manifested under functional disturbances. Thus, the efficiency of this mechanism in the synaptic region selectively increases in rats under hindlimb unloading, and decreases in the case of dystrophin deficit in mdx mice. The latter suggests that dystrophin is a molecular component essential for local hyperpolarization of the end-plate membrane.

Functional and immunocytochemical characterization of the creatine transporter in rat hippocampal neurons

Creatine uptake by neurons requires a specific creatine transporter (CRT). The purpose of the present work was to investigate the activity and localization of the CRT in primary cultures of hippocampal neurons obtained from 18-day rat embryos. Creatine uptake increased as the neurons differentiated in culture. Immunofluorescence microscopy showed most of the CRT was associated with dendrites, although some CRT was present in axons and axon terminals. Neurons contained high levels of Na(+)-dependent creatine transport activity (K(m) = 45.5 μM; V(max), 1719 pmol creatine/min/mg protein) which was inhibited by competitive inhibitors of the CRT. The IC(50) for guanidinoacetate, a precursor of creatine, was 712 μM, ∼ 15-fold higher than the K(m) for creatine. Incubation of neurons with 1 mM creatine resulted in the accumulation of high levels of creatine which affected the V(max) but not the K(m) for creatine transport. The rate of creatine release from neurons increased in the absence of Na(+) showing the importance of the electrochemical gradient for creatine retention. This is the first detailed study of the CRT in neurons and identifies primary cultures of rat hippocampal neurons as a good model for future studies of the CRT in relation to the effects of creatine on neuronal function and viability.

Characterization and catalytic behavior of Na-W-Mn-Zr-S-P/SiO 2 prepared by different methods in oxidative coupling of methane

Na-W-Mn-Zr-S-P/SiO 2 catalysts for oxidative coupling of methane (OCM) were prepared by incipient wetness impregnation, sol-gel and mixture slurry methods. The catalyst prepared by mixture slurry method showed the best catalytic performance among all samples. In addition, the effects of different addition sequences of Na, W, Mn, Zr, S and P on the catalytic performance were studied. The absence of Na before the addition of Mn and Zr in the catalysts preparation depressed the formation of the active phases of Mn 2O 3 and ZrO 2 and decreased the activities of the catalysts significantly.

Nigericin-mediated liposome loading of topotecan: Is nigericin a potential drug release regulator?

Topotecan was loaded into large unilamellar vesicles using nigericin-generated pH gradient or with triethylammonium (TA) ion gradient. Despite that in both loading methods, the encapsulated counter ion was 5-sulfosalicylate (5ssa), the resultant formulations exhibited distinct properties. In NaCl-containing release buffer, vesicles prepared with nigericin/Na + system could release topotecan at a faster rate than 5ssa–TA vesicles, whereas in the absence of Na +, there was no difference in the drug release kinetics. In plasma, both formulations could prolong the circulation halftime of topotecan, but 5ssa–TA vesicles were more able to stabilize the encapsulated topotecan, as evidenced by the increased t 1/2 and decreased conversion of lactone to carboxylate form. However, the improved drug retention did not mean the elevated safety and efficacy. In L1210 ascitic tumor model, the administration of 5ssa–TA vesicles at 10 mg/kg induced the early death of ∼60% mice at 6–7 days. In contrast, the treatment with nigericin/Na + vesicles at the same dose level resulted in a mean survival time of ∼18.0 days, ∼1.38-fold of that of free topotecan. In addition, the formulation was safer than free topotecan. The results indicated that nigericin could be used as release regulator to optimize drug release kinetics, resulting in safe and efficacious liposome-based topotecan formulation. The results were promising since no liposome topotecan formulations with reduced toxicity were reported.

Extracellular Allosteric Regulatory Subdomain within the γ Subunit of the Epithelial Na+ Channel

The activity of the epithelial Na(+) channel (ENaC) is modulated by Na(+) self-inhibition, a down-regulation of the open probability of ENaC by extracellular Na(+). A His residue within the extracellular domain of gammaENaC (gammaHis(239)) was found to have a critical role in Na(+) self-inhibition. We investigated the functional roles of residues in the vicinity of this His by mutagenesis and analyses of Na(+) self-inhibition responses in Xenopus oocytes. Significant changes in the speed and magnitude of Na(+) self-inhibition were observed in 16 of the 47 mutants analyzed. These 16 mutants were distributed within a 22-residue tract. We further characterized this scanned region by examining the accessibility of introduced Cys residues to the sulfhydryl reagent MTSET. External MTSET irreversibly increased or decreased currents in 13 of 47 mutants. The distribution patterns of the residues where substitutions significantly altered Na(+) self-inhibition or/and conferred sensitivity to MTSET were consistent with the existence of two helices within this region. In addition, single channel recordings of the gammaH239F mutant showed that, in the absence of Na(+) self-inhibition and with an increased open probability, ENaCs still undergo transitions between open and closed states. We conclude that gammaHis(239) functions within an extracellular allosteric regulatory subdomain of the gamma subunit that has an important role in conferring the response of the channel to external Na(+).

Substrate specificity of Rhbg: ammonium and methyl ammonium transport

Rhbg is a nonerythroid membrane glycoprotein belonging to the Rh antigen family. In the kidney, Rhbg is expressed at the basolateral membrane of intercalated cells of the distal nephron and is involved in NH4+ transport. We investigated the substrate specificity of Rhbg by comparing transport of NH3/NH4+ with that of methyl amine (hydrochloride) (MA/MA+), often used to replace NH3/NH4+, in oocytes expressing Rhbg. Methyl amine (HCl) in solution exists as neutral methyl amine (MA) in equilibrium with the protonated methyl ammonium (MA+). To assess transport, we used ion-selective microelectrodes and voltage-clamp experiments to measure NH3/NH4+- and MA/MA+-induced intracellular pH (pH(i)) changes and whole cell currents. Our data showed that in Rhbg oocytes, NH3/NH4+ caused an inward current and decrease in pH(i) consistent with electrogenic NH4+ transport. These changes were significantly larger than in H2O-injected oocytes. The NH3/NH4+-induced current was not inhibited in the presence of barium or in the absence of Na+. In Rhbg oocytes, MA/MA+ caused an inward current but an increase (rather than a decrease) in pH(i). MA/MA+ did not cause any changes in H2O-injected oocytes. The MA/MA+-induced current and pH(i) increase were saturated at higher concentrations of MA/MA+. Amiloride inhibited MA/MA+-induced current and the increase in pH(i) in oocytes expressing Rhbg but had no effect on control oocytes. These results indicate that MA/MA+ is transported by Rhbg but differently than NH3/NH4+. The protonated MA+ is likely a direct substrate whose transport resembles that of NH4+. Transport of electroneutral MA is also enhanced by expression of Rhbg.

Structure-Function Relations in Oxaloacetate Decarboxylase Complex. Fluorescence and Infrared Approaches to Monitor Oxomalonate and Na+ Binding Effect

BackgroundOxaloacetate decarboxylase (OAD) is a member of the Na+ transport decarboxylase enzyme family found exclusively in anaerobic bacteria. OAD of Vibrio cholerae catalyses a key step in citrate fermentation, converting the chemical energy of the decarboxylation reaction into an electrochemical gradient of Na+ ions across the membrane, which drives endergonic membrane reactions such as ATP synthesis, transport and motility. OAD is a membrane-bound enzyme composed of α, β and γ subunits. The α subunit contains the carboxyltransferase catalytic site.Methodology/Principal FindingsIn this report, spectroscopic techniques were used to probe oxomalonate (a competitive inhibitor of OAD with respect to oxaloacetate) and Na+ effects on the enzyme tryptophan environment and on the secondary structure of the OAD complex, as well as the importance of each subunit in the catalytic mechanism. An intrinsic fluorescence approach, Red Edge Excitation Shift (REES), indicated that solvent molecule mobility in the vicinity of OAD tryptophans was more restricted in the presence of oxomalonate. It also demonstrated that, although the structure of OAD is sensitive to the presence of NaCl, oxomalonate was able to bind to the enzyme even in the absence of Na+. REES changes due to oxomalonate binding were also observed with the αγ and α subunits. Infrared spectra showed that OAD, αγ and α subunits have a main component band centered between 1655 and 1650 cm−1 characteristic of a high content of α helix structures. Addition of oxomalonate induced a shift of the amide-I band of OAD toward higher wavenumbers, interpreted as a slight decrease of β sheet structures and a concomitant increase of α helix structures. Oxomalonate binding to αγand α subunits also provoked secondary structure variations, but these effects were negligible compared to OAD complex.ConclusionOxomalonate binding affects the tryptophan environment of the carboxyltransferase subunit, whereas Na+ alters the tryptophan environment of the β subunit, consistent with the function of these subunits within the enzyme complex. Formation of a complex between OAD and its substrates elicits structural changes in the α-helical as well as β-strand secondary structure elements.