Characterization of the complex formation of 1,6-anhydro-β-maltose and potassium ions using NMR spectroscopy and single-crystal X-ray crystallography

The formation of a complex between 1,6-anhydro-β-maltose and potassium ions was characterized using ¹H, ¹³C and ³⁹K NMR spectroscopy and single-crystal X-ray crystallography. In the NMR study, the spin-lattice relaxation times (T₁) of C1, C3, C5, C6, and C5′ significantly decreased in the presence of potassium ions, and ³⁹K-T₁ also decreased in the presence of 1,6-anhydro-β-maltose, indicating complex formation. In a crystal, both 8- and 9-coordination structures, corresponding to the distorted capped pentagonal bipyramidal structure and the capped hexagonal bipyramidal structure, respectively, were identified. A potassium ion was positioned in the center of each bipyramidal structure.     

1H-NMR study of GM2 ganglioside: evidence that an interresidue amide-carboxyl hydrogen bond contributes to stabilization of a preferred conformation

Several properties of the exchangeable amide protons of the ganglioside G_M2 were studied in detail by¹H-NMR spectroscopy in fully deuterated dimethylsulfoxide [²H₆]DMSO/2% H₂O, and compared with data obtained for the simpler constituent glycosphingolipids G_A2 and G_M3. In addition to chemical shifts,³ J _2,HN coupling constants, and temperature shift coefficients, the kinetics of NH/²H chemical exchange were examined by following the disappearance of the amide resonances in [²H₆]DMSO/2%²H₂O. The results included observation of an increase in half-life of theN-acetylgalactosamine acetamido HN by more than an order of magnitude in G_M2 compared to G_A2, attributable to the presence of the additionalN-acetylneuraminic acid residue. Additional one-dimensional dipolar cross relaxation experiments were also performed on nonexchangeable protons of G_M2. The results of all of these experiments support a three-dimensional model for the terminal trisaccharide in which a hydrogen bond is formed between theN-acetylgalactosamine acetamido NH and theN-acetylneuraminic acid carboxyl group. The interaction is proposed to be of the -acceptor type, a possibility which has not yet been explored in the literature on carbohydrates. The proposed model is discussed in comparison with that of Sabesanet al. (1984,Can J Chem 62: 1034–45), and the models of G_M1 proposed more recently by Acquottiet al. (1990,J Am Chem Soc 112:7772–8) and Scarsdaleet al. (1990,Biochemistry 29:9843–55).     

The vicissitudes of the pacemaker current <Emphasis Type="Italic">I</Emphasis><Subscript>Kdd</Subscript> of cardiac purkinje fibers

Summary The mechanisms underlying the pacemaker current in cardiac tissues is not agreed upon. The pacemaker potential in Purkinje fibers has been attributed to the decay of the potassium current I _Kdd. An alternative proposal is that the hyperpolarization-activated current I _f underlies the pacemaker potential in all cardiac pacemakers. The aim of this review is to retrace the experimental development related to the pacemaker mechanism in Purkinje fibers with reference to findings about the pacemaker mechanism in the SAN as warranted. Experimental data and their interpretation are critically reviewed. Major findings were attributed to K⁺ depletion in narrow extracellular spaces which would result in a time dependent decay of the inward rectifier current I _K1. In turn, this decay would be responsible for a “fake” reversal of the pacemaker current. In order to avoid such a postulated depletion, Ba²⁺ was used to block the decay of I _K1. In the presence of Ba²⁺ the time-dependent current no longer reversed and instead increased with time and more so at potentials as negative as −120 mV. In this regard, the distinct possibility needs to be considered that Ba²⁺ had blocked I _Kdd (and not only I _K1). That indeed this was the case was demonstrated by studying single Purkinje cells in the absence and in the presence of Ba²⁺. In the absence of Ba²⁺, I _Kdd was present in the pacemaker potential range and reversed at E _K. In the presence of Ba²⁺, I _Kdd was blocked and I _f appeared at potentials negative to the pacemaker range. The pacemaker potential behaves in a manner consistent with the underlying I _Kdd but not with I _f. The fact that I _f is activated on hyperpolarization at potential negative to the pacemaker range makes it suitable as a safety factor to prevent the inhibitory action of more negative potentials on pacemaker discharge. It is concluded that the large body of evidence reviewed proves the pacemaker role of I _Kdd (but not of I _f) in Purkinje fibers.     

Direct measurement of increased myocardial cellular23Na NMR signals in perfused guinea-pig heart induced by dihydroouabain and grayanotoxin-I

The effects of the cardiac glycoside dihydroouabain (DHO), and the ericaceous toxin grayanotoxin-I (GTX-I) on myocardial cellular sodium (Na_i) concentrations were investigated using sodium-23 nuclear magnetic resonance (²³Na NMR) spectroscopy at 30°C in isolated perfused guinea-pig hearts. The Na_i NMR signals from perfused Langendorff heart preparations were obtained by the modified inversion recovery (IR) method based on the previous observation that the spin-lattice relaxation time (T₁) of the Na_i (25 or 34 msec at 8.46 Tesla (T)) is much faster than that of extracellular sodium (64 msec at 9.4 T). Na_i was estimated from the calibration curve of the frequency area of the²³Na NMR FT spectra plotted against the standard Na concentration. The Na_i concentration of the heart increased concomitantly with the positive inotropic effects (PIE) of DHO, GTX-I and monensin (MON). The cumulative sequential addition of DHO (5×10^–6 M), GTX-I (7×10^–8 M) and MON (5×10^–6 M), each of which alone induced no appreciable PIE, produced a 22% elevation in Na_i concentration relative to that of the control (100%) accompanying a PIE of 44%. The mechanism of this Na_i elevation induced by combinational addition of DHO, GTX-I and MON may be mediated as follows: GTX-I increases the net Na-influxvia Na⁺ channels; DHO inhibits the pumping out of Na⁺ from the cell; and MON transports external Na⁺ into the cell, acting as a sodium ionophore. Consequently, these drugs act synergistically to increase the Na_i, thereby increasing the intracellular Ca²⁺ concentrationvia Na⁺–Ca²⁺ exchange.     

1H NMR Relaxometry in Natural Humous Soil Samples: Insights in Microbial Effects on Relaxation Time Distributions

¹H NMR relaxometry is applied for the investigation of pore size distributions in geological substrates. The transfer to humous soil samples requires the knowledge of the interplay between soil organic matter, microorganisms and proton relaxation. The goal of this contribution is to give first insights in microbial effects in the ¹H NMR relaxation time distribution in the course of hydration of humous soil samples. We observed the development of the transverse relaxation time distribution of the water protons after addition of water to air dried soil samples. Selected samples were treated with cellobiose to enhance microbial activity. Besides the relaxation time distribution, the respiratory activity and the total cell counts were determined as function of hydration time. Microbial respiratory activities were 2–15 times higher in the treated samples and total cell counts increased in all samples from 1×10⁹ to 5×10⁹ cells g⁻¹ during hydration. The results of ¹H NMR relaxometry showed tri-, bi- and mono-modal relaxation time distributions and shifts of peak relaxation times towards lower relaxation times of all investigated soil samples during hydration. Furthermore, we found lower relaxation times and merging of peaks in soil samples with higher microbial activity. Dissolution and hydration of cellobiose had no detectable effect on the relaxation time distributions during hydration. We attribute the observed shifts in relaxation time distributions to changes in pore size distribution and changes in spin relaxation mechanisms due to dissolution of organic and inorganic substances (e.g. Fe³⁺, Mn²⁺), swelling of soil organic matter (SOM), production and release of extracellular polymeric substances (EPS) and bacterial association within biofilms.     

A suite of Mathematica notebooks for the analysis of protein main chain 15N NMR relaxation data

A suite of Mathematica notebooks has been designed to ease the analysis of protein main chain ¹⁵N NMR relaxation data collected at a single magnetic field strength. Individual notebooks were developed to perform the following tasks: nonlinear fitting of ¹⁵N-T ₁ and -T ₂ relaxation decays to a two parameter exponential decay, calculation of the principal components of the inertia tensor from protein structural coordinates, nonlinear optimization of the principal components and orientation of the axially symmetric rotational diffusion tensor, model-free analysis of ¹⁵N-T ₁, -T ₂, and {¹H}–¹⁵N NOE data, and reduced spectral density analysis of the relaxation data. The principle features of the notebooks include use of a minimal number of input files, integrated notebook data management, ease of use, cross-platform compatibility, automatic visualization of results and generation of high-quality graphics, and output of analyses in text format.L. Spyracopoulos is an AHFMR Medical Research Senior Scholar     

Different Ks values for hydrogen of methanogenic bacteria and sulfate reducing bacteria: An explanation for the apparent inhibition of methanogenesis by sulfate

Desulfovibrio vulgaris (Marburg) and Methanobrevibacter arboriphilus (AZ) are anaerobic sewage sludge bacteria which grow on H₂ plus sulfate and H₂ plus CO₂ as sole energy sources, respectively. Their apparent K_s values for H₂ were determined and found to be approximately 1 M for the sulfate reducing bacterium and 6 M for the methanogenic bacterium. In mixed cell suspensions of the two bacteria (adjusted to equal V _max) the rate of H₂ consumption by D. vulgaris was five times that of M. arboriphilus, when the hydrogen supply was rate limiting. The apparent inhibition of methanogenesis was of the same order as expected from the different K_s values for H₂. Difference in substrate affinities can thus account for the inhibition of methanogenesis from H₂ and CO₂ in sulfate rich environments, where the H₂ concentration is well below 5 M.     

1H NMR study of the interaction of bacteriophage λ Cro protein with the O R 3 operator

The 17 base pair operator O _R ³ oligonucleotide, which is the preferential binding site for the Cro repressor of phage , was studied by two-dimensional NMR spectroscopy. A sequential assignment procedure based on two-dimensional Nuclear Overhauser Effect (NOESY) and scalar coupling correlated (COSY) NMR spectroscopy, together with the knowledge of the oligodesoxynucleotide sequence, made it possible to assign the non-exhangeable base protons and the H1 and the H2-H2 sugar protons of the O _R ³ operator DNA. The pattern of the observed NOE connectivities is consistent with a right-handed helical DNA structure. The base and sugar proton assignments provide the necessary information for further studies of the O _R ³ operator — Cro repressor interaction.Abbreviations COSY correlated spectroscopy - FID free induction decay - NOE nuclear Overhauser effect - NOESY nuclear Overhauser effect spectroscopy - RD relaxation delay - TSP sodium 3-trimethylsilyl-(2,2,3,3-²H₄)propionate - EDTA sodium ethylendiamine tetraacetate     

Residue Specific Ribose and Nucleobase Dynamics of the cUUCGg RNA Tetraloop Motif by MNMR 13C Relaxation

The dynamics of the nucleobase and the ribose moieties in a 14-nt RNA cUUCGg hairpin-loop uniformly labeled with ¹³C and ¹⁵N were studied by ¹³C spin relaxation experiments. R₁, R_1ρ and the ¹³C-{¹H} steady-state NOE of C₆ and C_1′ in pyrimidine and C₈ and C_1′ in purine residues were obtained at 298 K. The relaxation data were analyzed by the model-free formalism to yield dynamic information on timescales of pico-, nano- and milli-seconds. An axially symmetric diffusion tensor with an overall rotational correlation time τ_c of 2.31±0.13 ns and an axial ratio of 1.35±0.02 were determined. Both findings are in agreement with hydrodynamic calculations. For the nucleobase carbons, the validity of different reported ¹³C chemical shift anisotropy values (Stueber, D. and Grant, D. M., 2002 J. Am. Chem. Soc. 124, 10539–10551; Fiala et al., 2000 J. Biomol. NMR 16, 291–302; Sitkoff, D. and Case, D. A., 1998 Prog. NMR Spectroscopy 32, 165–190) is discussed. The resulting dynamics are in agreement with the structural features of the cUUCGg motif in that all residues are mostly rigid (0.82 < S² < 0.96) in both the nucleobase and the ribose moiety except for the nucleobase of U7, which is protruding into solution (S² = 0.76). In general, ribose mobility follows nucleobase dynamics, but is less pronounced. Nucleobase dynamics resulting from the analysis of ¹³C relaxation rates were found to be in agreement with ¹⁵N relaxation data derived dynamic information (Akke et al., 1997 RNA 3, 702–709). Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

The trans labilization of cis-[PtCl2(13CH3NH2)2] by glutathione can be monitored at physiological pH by [1H,13C] HSQC NMR

In order to monitor the trans labilization of cisplatin at physiological pH we have prepared the complex cis-[PtCl₂(¹³CH₃NH₂)₂] and studied its interactions with excess glutathione in aqueous solution at neutral pH by two-dimensional [1H,13C] heteronuclear single-quantum correlation (HSQC) NMR spectroscopy. [1H,13C] HSQC spectroscopy is a good method for following the release of ¹³CH₃NH₂ but is not so good for characterizing the Pt species in solution. In the reaction of cisplatin with glutathione, Pt–S bonds are formed and Pt–NH₃ bonds are broken. The best technique for following the formation of Pt–S bonds of cisplatin is by UV spectroscopy. [1H,13C] HSQC spectroscopy is the best method for following the breaking of the Pt–N bonds. [1H,15N] HSQC spectroscopy is the best method for characterizing the different species in solution. However, the intensity of the peaks in the ¹⁵NH₃–Pt–S region, in [1H,15N] HSQC, reflects a balance between the formation of Pt–S bonds, which increases the signal intensity, and the trans labilization, which decreases the signal intensity. [1H,15N] HSQC spectroscopy and [1H,13C] HSQC spectroscopy are complementary techniques that should be used in conjunction in order to obtain the most accurate information on the interaction of platinum complexes with sulfur-containing ligands.     

Internal mobility of the ologonucleotide duplexes d(TCGCG)2 and d(CGCGCG)2 in aqueous solution from molecular dynamics simulations

Summary In this paper we present longitudinal relaxation times, order parameters and effective correlation times for the base and sugar carbons in both strands of the oligonucleotide duplexes d(TCGCG)₂ and d(CGCGCG)₂, as calculated from 400 ps molecular dynamics trajectories in aqueous solution. The model-free approach (Lipari and Szabo, 1982) was used to determine the amplitudes and time scales of the internal motion. Comparisons were made with NMR relaxation measurements (Borer et al., 1994). The order parameters could acceptably be reproduced, and the effective correlation times were found to be lower than the experimental estimates. Reasonable T₁ relaxation times were obtained in comparison with experiment for the nonterminal nucleosides. The T₁ relaxation times were found to depend mainly on the order parameters and overall rotational correlation time.Abbreviations MD molecular dynamics - CSA chemical shift anisotropy To whom correspondence should be addressed.     

Potentiometric and P NMR studies on inositol phosphates and their interaction with iron(III) ions

Potentiometric, conductometric and ³¹P NMR titrations have been applied to study interactions between myo-inositol hexakisphosphate (phytic acid), (±)-myo-inositol 1,2,3,5-tetrakisphosphate and (±)-myo-inositol 1,2,3-trisphosphate with iron(III) ions. Potentiometric and conductometric titrations of myo-inositol phosphates show that addition of iron increases acidity and consumption of hydroxide titrant. By increasing the Fe(III)/InsP₆ ratio (from 0.5 to 4) 3 mol of protons are released per 2 mol of iron(III). At first, phytates coordinate iron octahedrally between P2 and P1,3. The second coordination site represents P5 and neighbouring P4,6 phosphate groups. Complexation is accompanied with the deprotonation of P1,3 and P4,6 phosphate oxygens. At higher concentration of iron(III) intermolecular P–O–Fe–O–P bonds trigger formation of a polymeric network and precipitation of the amorphous Fe(III)–InsP₆ aggregates. ³¹P NMR titration data complement the above results and display the largest chemical shift changes at pD values between 5 and 10 in agreement with strong interactions between iron and myo-inositol phosphates. The differences in T₁ relaxation times of phosphorous atoms have shown that phosphate groups at positions 1, 2 and 3 are complexated with iron(III). The interactions between iron(III) ions and inositol phosphates depend significantly on the metal to ligand ratio and an attempt to coordinate more than two irons per InsP₆ molecule results in an unstable heterogeneous system.     

Single-channel K+ currents inDrosophila muscle and their pharmacological block

Summary Four types of nonvoltage-activated potassium channels in the body-wall muscles ofDrosophila third instar larvae have been identified by the patch-clamp technique. Using the inside-out configuration, tetraethylammonium (TEA). Ba²⁺, and quinidine were applied to the cytoplasmic face of muscle membranes during steady-state channel activation. The four channels could be readily distinguished on the basis of their pharmacological sensitivities and physiological properties. The K_ST channel was the only type that was activated by stretch. It had a high unitary conductance (100 pS in symmetrical 130/130mm KCl solution), was blocked by TEA (K _d 35mm), and was the most sensitive to Ba²⁺ (complete block at 10^–4 m). A Ca²⁺-activated potassium channel. K_CF 72pS (130/130mm KCl), was gated open at>10^–8 m Ca²⁺, was the least sensitive to Ba²⁺ (K _d of 3mm) and TEA (K _d of 100mm), and was not affected by quinidine. K₂ was a small conductance channel of 11 pS (130/2 KCl, pipette/bath), and was very sensitive to quinidine, being substantially blocked at 0.1mm. It also exhibited a half block at 0.3mm Ba²⁺ and 25mm TEA. A fourth channel type, K₃, was the most sensitive to TEA (half block<1mm). It displayed a partial block to Ba²⁺ at 10mm, but no block by 0.1mm quinidine. The blocking effects of TEA, Ba²⁺ and quinidine were reversible in all channels studied. The actions of TEA and Ba²⁺ appeared qualitatively different: in all four channels. TEA reduced the apparent unitary conductance, whereas Ba²⁺ decreased channel open probability.     

Unraveling protein dynamics through fast spectral density mapping

Spectral density mapping at multiple NMR field strengths is probably the best method to describe the dynamical behavior of a protein in solution through the analysis of ¹⁵N heteronuclear relaxation parameters. Nevertheless, such analyses are scarcely reported in the literature, probably because this method is excessively demanding in spectrometer measuring time. Indeed, when using n different magnetic fields and assuming the validity of the high frequency approximation, the discrete sampling of the spectral density function with 2n + 1 points needs the measurement of 3n ¹⁵N heteronuclear relaxation measurements (n R ₁, n R ₂, and n¹⁵N{¹H}NOEs). Based on further approximations, we proposed a new strategy that allows us to describe the spectral density with n + 2 points, with the measurement of a total of n + 2 heteronuclear relaxation parameters. Applied to the dynamics analysis of the protein p13 ^MTCP1 at three different NMR fields, this approach allowed us to divide by nearly a factor of two the total measuring time, without altering further results obtained by the “model free” analysis of the resulting spectral densities. Furthermore, simulations have shown that this strategy remains applicable to any low isotropically tumbling protein ( ns), and is valid for the types of motion generally envisaged for proteins.     

Pathways for K<Superscript>+</Superscript> Efflux in Isolated Surface and Crypt Colonic Cells. Activation by Calcium

K⁺-conductive pathways were evaluated in isolated surface and crypt colonic cells, by measuring ⁸⁶Rb efflux. In crypt cells, basal K⁺ efflux (rate constant: 0.24 ± 0.044 min⁻¹, span: 24 ± 1.3%) was inhibited by 30 mM TEA and 5 mM Ba²⁺ in an additive way, suggesting the existence of two different conductive pathways. Basal efflux was insensitive to apamin, iberiotoxin, charybdotoxin and clotrimazole. Ionomycin (5 μM) stimulated K⁺ efflux, increasing the rate constant to 0.65 ± 0.007 min⁻¹ and the span to 83 ± 3.2%. Ionomycin-induced K⁺ efflux was inhibited by clotrimazole (IC₅₀ of 25 ± 0.4 μM) and charybdotoxin (IC₅₀ of 65 ± 5.0 nM) and was insensitive to TEA, Ba²⁺, apamin and iberiotoxin, suggesting that this conductive pathway is related to the Ca²⁺-activated intermediate-conductance K⁺ channels (IK_ca). Absence of extracellular Ca²⁺ did neither affect basal nor ionomycin-induced K⁺ efflux. However, intracellular Ca²⁺ depletion totally inhibited the ionomycin-induced K⁺ efflux, indicating that the activation of these K⁺ channels mainly depends on intracellular calcium liberation. K⁺ efflux was stimulated by intracellular Ca²⁺ with an EC₅₀ of 1.1 ± 0.04 μM. In surface cells, K⁺ efflux (rate constant: 0.17 ± 0.027 min⁻¹; span: 25 ± 3.4%) was insensitive to TEA and Ba²⁺. However, ionomycin induced K⁺ efflux with characteristics identical to that observed in crypt cells. In conclusion, both surface and crypt cells present IK_Ca channels but only crypt cells have TEA- and Ba²⁺-sensitive conductive pathways, which would determine their participation in colonic K⁺ secretion.     

Simple device to monitor aerobic biotransformations by in situ 1H-NMR

A simple device for taking in situ proton NMR measurements in ¹H₂O is described. This allows aeration of reactions in a 10 mm diameter NMR tube without modifying the magnet or the probe head. With this device, aerobic biotransformations can be monitored in the NMR-tube placed in the spectrometer. It allows in situ analyses of the transformations, separating the aeration period temporally from the measurement time, not unlike traditional Warburg respiratory experiments. Two reactions determining kinetic and stoichieometric parameters: (i) a biotransformation by a growing Pseudomonas putida culture and (ii) l-phenylalanine oxidation catalysed by l-amino acid oxidase [E.C. 1.4.3.2]; both incubations were contained in the magnet.     

Lithium nuclear magnetic resonance measurements in halotolerant bacterium Ba1

Measurements of relaxation times T₁ and T₂, were carried out on a high-salt- and low-salt-grown bacterial pellets of halotolerant bacterium B_a1. In our measurements, T₁ ? T₂ and both were frequency-independent. In the high-salt-grown pellet the relaxation time values were much shorter than in the case of low-salt growth medium. Intensity measurements show that only 55% of the lithium in the high-salt pellet is detected; for the low-salt pellet almost all the lithium is detected. Growth measurements were carried out on the B_a1. It is suggested that there is some form of adaptation of the bacteria to the growth medium. The adaptation is reflected in the lithium NMR results.     

31P NMR relaxation measurements of the phosphate backbone of a double stranded hexadeoxynucleotide in solution: determination of the chemical shift anisotropy

The five phosphates of the deoxynucleotide d(CpGpTpApCpG)₂ have been assigned by two-dimensional heteronuclear NMR spectroscopy. The chemical shift anisotropy and correlation time of each phosphate group has been determined from measurements of the spin-lattice, spin-spin relaxation rate constants and the ³¹P-{¹H} nuclear Overhauser enhancement (NOE) at three magnetic field strengths (4.7 T, 9.4 T, and 11.75 T) and two temperatures (288 K and 298 K). As expected, the relaxation data require two mechanisms to account for the observed rate constants, i.e. dipole-dipole and chemical shift anisotropy. At 9.4 T and 11.75 T, the latter mechanism dominates the relaxation, leading to insignificant NOE intensities. The correlation time, chemical shift anisotropy and effective P-H distance were obtained from least-squares fitting to the data. Comparison of the fitted value for the correlation time with that obtained from ¹H measurements shows that the molecule behaves essentially as rigid rotor on the nanosecond timescale. Large amplitude motions observed in long segments of DNA are due to bending motions that do not contribute significantly to relaxation in short oligonucleotides.Abbreviations CSA chemical shift anisotropy - NOE nuclear Overhauser enhancement Offprint requests to: A. N. Lane     

Vasotocin has the potential to inhibit basolateral Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-pump current across isolated skin of tree frog in vitro<Emphasis Type="Italic">,</Emphasis> via its V<Subscript>2</Subscript>-type receptor/cAMP pathway

Adult frog skin transports Na⁺ from the apical to the basolateral side across the skin. Antidiuretic hormone (ADH) is involved in the regulation of Na⁺ transport in both mammals and amphibians. We investigated the effect of arginine vasotocin (AVT), the ADH of amphibians, on the short-circuit current (SCC) across intact skin and on the basolateral Na⁺/K⁺-pump current across apically nystatin-permeabilized skin of the tree frog, Hyla japonica, in which the V₂-type ADH receptor is expressed in vitro. In intact skin, 1 pM AVT had no effect on the SCC, but 10 nM AVT was sufficient to stimulate the SCC since 10 nM and 1 μM of AVT increased the SCC 3.2- and 3.4-fold, respectively (P > 0.9). However, in permeabilized skin, AVT (1 μM) decreased the Na⁺/K⁺-pump current to 0.79 times vehicle control. Similarly, 500 μM of 8Br-cAMP increased the SCC 3.2-fold, yet 1 mM of 8Br-cAMP decreased the Na⁺/K⁺-pump current to 0.76 times vehicle control. Arachidonic acid (10⁻⁵ M) tended to decrease the Na⁺/K⁺-pump current. To judge from these in vitro experiments, AVT has the potential to inhibit the basolateral Na⁺/K⁺-pump current via the V₂-type receptor/cAMP pathway in the skin of the tree frog.     

ATP Synthases With Novel Rotor Subunits: New Insights into Structure,Function and Evolution of ATPases

ATPases with unusual membrane-embedded rotor subunits were found in both F₁F₀ and A₁A₀ ATP synthases. The rotor subunit c of A₁A₀ ATPases is, in most cases, similar to subunit c from F₀. Surprisingly, multiplied c subunits with four, six, or even 26 transmembrane spans have been found in some archaea and these multiplication events were sometimes accompanied by loss of the ion-translocating group. Nevertheless, these enzymes are still active as ATP synthases. A duplicated c subunit with only one ion-translocating group was found along with “normal” F₀ c subunits in the Na⁺ F₁F₀ ATP synthase of the bacterium Acetobacterium woodii. These extraordinary features and exceptional structural and functional variability in the rotor of ATP synthases may have arisen as an adaptation to different cellular needs and the extreme physicochemical conditions in the early history of life.