Sodium exchange between two sites. The binding of sodium to halotolerant bacteria.

The longitudinal and transverse relaxation curves of sodium undergoing exchange between two sites are presented. When the two sites are 'bound' and 'free' sodium respectively, the relaxation curves are, in general, not exponential. It is shown that in some cases only one exponential decay could be detected experimentally though the true decay curve is much more complicated. In such cases where the population of 'free' and 'bound' sodium are equal, only 40-70% of the total intensity would be detected, depending on the lifetime of sodium in the two sites. It is also shown that the fast exchange approximation, usually applied in the interpretation of sodium relaxation curves, might lead to wrong conclusions. Measurements of sodium relaxation times in halotolerant bacteria show that T1 and T2 are different and frequency-dependent. The intensity of the sodium sugnal is 40% of the total sodium concentration. It was possible to simulate the relaxation behaviour and intensity measurements by applying the following model. There are three types of sodium, the extracellular sodium (A) which exchanges with part of the intracellular sodium (B) and a fraction (C) which is bound but does not exchange with the extracellular sodium. It was possible to estimate the physical properties of sodium at site B. The quadrupole coupling constant (e2qQ/H)B = 9.10(6) rad/s, the correlation time taucB = 5.5.10(-7) s and the lifetime of sodium at site B, tauB = 6.10(-4) s.

Nuclear magnetic resonance studies of Ba1 bacterium and some model systems.

Lithium NMR relaxation times of some model systems and E. coli cells in high LiCl concentration were measured. The lithium NMR relaxation times were compared to the relaxation times in the holotolerant bacterium Ba1 (Goldberg, M., Risk, M. and Gilboa, H. (1983) Biochim. Biophys. Acta 763, 35-40). Complementary studies of the water protons NMR relaxation times were carried out. It is suggested that the lithium in the H.S. Ba1 bacterium is occulated in small pores of the cell envelope.

Hydrogen bonds in the tripeptide Pro-Leu-Gly-NH2. 17O and 1H studies.

17O-NMR measurements of labeled Pro-Leu-Gly-NH2 were carried out at different pH levels and in mixed solvents of water/acetonitrile. Complementary studies of the amide protons were carried out in acetonitrile-d3. Only the prolyl C = 17O group was sensitive to the pH level. Protonation of the amine group resulted in an upfield chemical shift of 18 ppm. The chemical shifts of each of the three oxygen sites was sensitive to the ratio water:acetonitrile. Solvent composition dependence of the chemical shift and linewidth suggests that the prolyl C = 17O is involved in intramolecular hydrogen bond formation when Pro-Leu-Gly-NH2 is dissolved in acetonitrile, while in water there is no intramolecular H bond.

Nuclear magnetic resonance studies of cesium-133 in the halophilic halotolerant bacterium Ba1. Chemical shift and transport studies.

Ba1 bacteria (Halomonas israelensis) were grown on different salt concentrations 0.2-4 M. When the cells were transferred to a medium containing 25 mM CsCl without potassium there was an uptake of cesium by the cells. The intracellular cesium signal was shifted from the cesium signal in the medium without the use of a shift reagent. The shift was depended on the salt concentration in the growth medium. The intracellular cesium shift showed a much smaller dependence on the concentration of salts in the medium than the extracellular cesium; the same results were detected for cells grown on a medium containing 25 mM CsCl. The cesium transport through the cell membrane was mostly by active transport. The cesium concentration in the cell was higher than that of the medium, approximately 100 mM intracellular concentration compared to 25 mM in the medium. The first order constants for influx or efflux of cesium were from 2 x 10(-4) and up to 24 x 10(-4)/min for the different medium concentrations.

Molecular motions and phase transitions. NMR relaxation times studies of several lecithins.

The spin-lattice relaxation time, T1, and the dipolar energy relaxation time, TD, were measured as a function of temperature. The materials studied were samples of anhydrous L-dipalmitoyl lecithin, DL-dipalmitoyl lecithin, L-dimyristoyl lecithin, DL-dimyristoyl lecithin and their monohydrates, and of anhydrous egg yolk lecithin. It is shown that TD is a much more sensitive parameter than T1 for the determination of the Chapman phase transition. Comparison between T1 and TD provides information about new types of slow molecular motions below and above the phase transition temperature. It is suggested that the relaxation mechanisms for T1 and TD in the gel phase are governed by segmental motion in the phospholipid molecule. A new metastable phase was detected in dimyristoyl lecithin monohydrates. This phase could only be detected from the dipolar energy relaxation times.

13C NMR study of the interrelation between synthesis and uptake of compatible solutes in two moderately halophilic eubacteria. Bacterium Ba1 and Vibro costicola.

The synthesis and uptake of intracellular organic osmolytes (compatible solutes) were studied with the aid of natural abundance 13C NMR spectroscopy in two unrelated, moderately halophilic eubacteria: Ba1 and Vibrio costicola. In minimal media containing 1 M NaCl, both microorganisms synthesized the cyclic amino acid, 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (trivial name, ectoine) as the predominant compatible solute, provided that no glycine betaine was present in the growth medium. When, however, the minimal medium was supplemented with glycine betaine or the latter was a component of a complex medium, it was transported into the cells and the accumulating glycine betaine replaced the ectoine. In Ba1, grown in a defined medium containing glucose as the single carbon source, ectoine could only be detected if the NaCl concentration in the medium was higher than 0.6 M; the ectoine content increased with the external salt concentration. At NaCl concentrations below 0.6 M, alpha,alpha-trehalose was the major organic osmolyte. The concentration of ectoine reached its peak during the exponential phase and declined subsequently. In contrast, the accumulation of glycine betaine continued during the stationary phase. The results presented here indicate that, at least in the two microorganisms studied, ectoine plays an important role in haloadaptation.

19F NMR magnetization transfer between 5-FBAPTA and its complexes. An alternative means for measuring free Ca2+ concentration, and detection of complexes with protein in erythrocytes.

The 19F NMR Ca(2+)-indicator molecule 5,5'-difluoro-1,2-bis(o- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (5-FBAPTA) was used in a procedure that enhances its range of applications to measuring free Ca2+ concentrations in buffer solutions and human erythrocytes. Even if the signal from the Ca-5-FBAPTA complex was not visible, the concentration of the complex could be calculated from saturation transfer spectra. This was demonstrated with well characterized buffer solutions in vitro and shown to also apply to concentrated haemolysates. The analysis required a precise estimate of the dissociation rate constant of the complex; this was found to be 295/s at 37 degrees C and the corresponding association rate constant was 4.1 x 10(8) L/mol/s. These values differ from those obtained previously in different buffer conditions and by two different NMR methods. A series of spectra were acquired from haemolysates containing 5-FBAPTA, in which saturating irradiation was applied at a frequency that was progressively off-set from the carrier frequency. Saturation transfer to the free 5-FBAPTA was seen from irradiation at frequencies different from that of Ca-5-FBAPTA, thus suggesting the presence of complexes with proteins.

The determination of intracellular sodium concentration in human red blood cells: nuclear magnetic resonance measurements.

The intracellular sodium concentration and intracellular volume of human red blood cells were determined from 23Na and 1H NMR spectra. It is shown that sodium dissolved in the intracellular water has a concentration higher than that previously published. The intracellular sodium concentration measured was 11.4 +/- 3.1 mM. A comparison of different NMR methods used to determine sodium concentration is given.

Use of 23Na nuclear magnetic resonance spectroscopy to determine the true intracellular concentration of free sodium in a halophilic eubacterium.

We present new data obtained by 23Na nuclear magnetic resonance spectroscopy, which can distinguish free intracellular sodium from cell-bound sodium, showing that the intracellular concentration of Na+ the halophilic eubacterium Vibrio costicola is only 5 to 20% of that in the extracellular medium. Previous methods could not distinguish free intracellular Na+ from that bound to cell structures, and it was believed that in halophilic eubacteria the total monovalent cation concentration inside matched that of the NaCl outside. Information obtained by the newer technology raises fundamental questions about the ways in which these organisms and others which live in hypersaline environments function and cope with osmotic stress.