39K, 23Na, and 31P NMR studies of ion transport in Saccharomyces cerevisiae.

Ogino, Takashi; Hollander, Jan A. den; Shulman, Robert G.

doi:10.1073/pnas.80.17.5185

Cited by 77 publications

(46 citation statements)

References 23 publications

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“…Until the 23Na NMR method was applied to the Na' analyses in cells, analyses were restricted to the cellassociated ions, and the bound or attached sodium content was not distinguished (Ogino et al, 1983;Gilboa et al, 1991). According to our analyses, the concentrations of free and bound sodium in the cytosol were 0.13-0.14 and 0.23-0-26 pmol (mg protein)-', respectively.…”

Section: Discussionmentioning

confidence: 99%

23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

et al. 1995

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23Na NMR spectroscopy was used to determine free Na+ concentrations in a halotolerant bacterium, Brewibacterium sp., and Escherichia coli. The internal Na+ concentration of both strains depended little on the growth phases and was unchanged after 5 d storage at 2 O C . In Brewibacterium sp. the level of intracellular sodium increased gradually at higher extracellular NaC! concentrations in both the presence and absence of yeast extract in the growth medium. E. coli cells accumulated a higher concentration of free Na+ than those of Brewibacterium sp. The change of Na+ concentration in both strains was inverse to that of growth rate. When appropriate amounts of osmoprotectants (proline, glycine betaine, or y-aminobutyrate) were added with the NaCI, internal free Na+ levels in Brewibacterium sp. were lowered, but those of E. coli were unchanged. While addition of KCI to medium containing NaCl increased the intracellular level of free Na+, the total sodium concentration in the cells remained unchanged, indicating that sodium that had been bound or attached was made free in the cytosol. In Brewibacterium sp. grown in the presence of 0.5 M NaCI, free and bound sodium concentrations in the cytosol were estimated to be 014 and 0-23 pmol (mg protein)-', respectively. As a result, visibility by 23Na NMR was 38%.

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Section: Discussionmentioning

confidence: 99%

23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

et al. 1995

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“…The observation of separate signals for intra-and extracellular Na+ and K+ as shown in Figs. 2 and 3 enabled us to determine directly their NMR visibility in RBC without physically isolating the intra-and extracellular compartments by inducing ion fluxes and comparing the changes in the NMR signals of intra-and extracellular Na" and K+ (11). In this experiment, RBC were resuspended into a Na'-free choline buffer at 250C and an ionophore, gramicidin (15 Na' were plotted against those of the extracellular Na' (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…A with our home-built solenoidal coil probe (11). During the NMR measurements, the cells were oxygenated and gently mixed with a propeller driven by an air turbine.…”

Section: Methodsmentioning

confidence: 99%

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23Na and 39K NMR studies of ion transport in human erythrocytes.

Ogino¹,

Shulman²,

Avison³

et al. 1985

Proc. Natl. Acad. Sci. U.S.A.

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Ion transport in human erythrocytes was studied by 23Na and 39K NMR with an anionic paramagnetic shift reagent, Dy(P3O10)7-. The intra-and extracellular 23Naand 39K NMR signals were well separated (over 10 ppm) at 5 mM concentration of the shift reagent. The NMR visibility of the intracellular Na' and K+ was determined to be 100% in human and duck erythrocytes. The intracellular ion concentrations were 8.1 + 0.8 mM Na+ (n = 7) and 110 ± 12 mM K+ (n = 4) for fresh human erythrocytes. The ouabain-sensitive net Na+ efflux was 1.75 ± 0.08 mmol/hr per liter of cells at 370C (n = 3). The gramicidin-induced ion transport in human erythrocytes was also studied by 23Na and 39K NMR or by simultaneous measurements of 23Na NMR and a V-selective electrode. The time courses of the Na+ and K+ transport induced by the ionophore were biphasic. The initial rapid fluxes were due to an exchange of Na+ for K+, which were found to occur with a 1:1 stoichiometry. The subsequent slow components were the net Na+ and K+ effluxes rate-limited by the Cl-permeability and accompanied by a reduction in cell volume. The Cl-permeability determined from the NMR measurements of these slow fluxes was 3.2 + 0.5 x 10-8 cm/sec at 250C (n = 4).Concentration gradients of Na+ and K+ across the erythrocyte (RBC) membrane are primarily maintained by an active transport of Na+ and K+ by the Na+,K+-pump (1). The kinetic properties of the Na+,K+-pump have been extensively studied in RBC. Alterations in Na+ fluxes with resultant abnormal intracellular Na+ concentration in RBC have been suggested to exist in a variety of disease processes, such as essential hypertension (2), affective disorders (3), myotonic dystrophy (4), and hyperthyroidism (5). Conventional methods of analysis for intracellular ions such as flame photometry and radioisotope tracer techniques have the disadvantage of requiring time-consuming destructive methods to achieve physical separation of intraand extracellular compartments. Furthermore, there are uncertainties associated with the possibility of nonspecific binding of ions to the cell membrane and of ion fluxes occurring during the separation procedure. Clearly, a nondestructive and noninvasive method would be welcome.NMR spectroscopy offers such an approach. The recent development of anionic paramagnetic shift reagents (SRs) (6, 7) has made it possible to resolve the intra-and extracellular NMR signals of 23Na and 39K in cells and tissues. This approach has been applied to measure intracellular Na+ (7,8) and K+ (9) concentrations and to study the physical state of intracellular Na+ in RBC (10). However, no studies of ion transport in RBC have been reported, even though the NMR method would have advantages in the study of ion fluxes (11).The present study demonstrates the suitability of NMR spectroscopy with the paramagnetic SR Dy(P3010)7-for measuring Na' and K+ fluxes as well as ion concentrations in human RBC. EXPERIMENTAL Blood was drawn from healthy donors into a syringe containing sodium heparin (10 units/ml). The RBC wer...

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“…1 (spectra C and D) showed that after lysis of the cells, there was, with time, a progressive downfield shift in the resonance attributable to the external sodium ions. This timedependent downfield shift observed upon lysis of the cells is believed to be the result of hydrolysis of the shift reagent by the released intracellular phosphatases (26). Thus, in these studies, downfield shifts in the external sodium resonances were taken to be indicators of cell lysis.…”

Section: Methodsmentioning

confidence: 99%

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques

et al. 1988

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The active transport of sodium ions in live Acholesplasma laidlawii B cells and in lipid vesicles containing the (Na+-Mg2e)-ATPase from the plasma membrane of this microorganism was studied by 23Na nuclear magnetic resonance spectroscopic and 22Na tracer techniques, respectively. In live A. laidlawii B cells, the transport of sodium was an active process id which metabolic energy was harnessed for the extrusion of sodium ions against a concentration gradient. The process was inhibited by low temperatures and by the formation of gel state lipid in the plasma membrane of this organism. In reconstituted proteoliposomes containing the purified (Na+-Mg2e)-ATPase, the hydrolysis Of ATP was accompanied by the transport of sodium ions into the lipid vesicles, and the transport process was impaired by reagents known to inhibit ATPase activity. At the normal growth temperature (37°C), this transport process required a maximum of 1 mol of ATP per mol of sodium ion transported. Together, these results provide direct experimental evidence that the (Na'-Mg2+)-ATPase of the Acholeplasma laidlawii B membrane is the cation pump which maintains the low levels of intracellular sodium characteristic of this microorganism.The maintenance of low levels of intracellular sodium ions appears to be an almost universal requirement of living cells (38,39). In most cases this requirement is met by the use of specialized cation pumps which harness metabolic energy to transport sodium ions, usually against a steep concentration gradient (13,21,22). Furthermore, such ion pumps tend to be an integral part of the osmoregulatory mechanism of the cell, since the movement of cations like sodium is often coupled with the movement of water, protons, other cations (especially potassium), and sometimes amino acids and other permeants (15). In the case of mycoplasmas like Acholeplasma laidlawii B, a small cell wall-less procaryote containing a single limiting or plasma membrane, the functioning of ion pumps and the other parts of their osmoregulatory mechanism is critical to their viability (16,33). These cells are usually osmotically fragile and tend to swell and eventually lyse whenever the activity of the ion pumps is impaired, either directly by inhibitors or indirectly by interruption of the supply of metabolic energy (19,38). The small size and osmotic fragility of organisms like Acholeplasma laidlawii B have so far precluded the use of traditional nondestructive methods for observing sodium transport and monitoring their internal levels of sodium ions. However, with the recent discovery of somne paramagnetic shift reagents for 23Na nuclear magnetic resonance (NMR) spectroscopy (5, 13, 28-30), noninvasive monitoring of sodium transport and internal sodium is now feasible. In this report, we describe the use of NMR spectroscopy to characterize sodium transport in live A. laidlawii B cells.Previous studies (see references 16,17,18,35,36,37, and references cited thereit) have identified and characterized an Mg2+-dependent, Na+-stimulated ATPase activ...

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39K, 23Na, and 31P NMR studies of ion transport in Saccharomyces cerevisiae.

Cited by 77 publications

References 23 publications

23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

23Na NMR spectroscopy of free Na+ in the halotolerant bacterium Brevibacterium sp. and Escherichia coli

23Na and 39K NMR studies of ion transport in human erythrocytes.

Characterization of sodium transport in Acholeplasma laidlawii B cells and in lipid vesicles containing purified A. laidlawii (Na+-Mg2+)-ATPase by using nuclear magnetic resonance spectroscopy and 22Na tracer techniques

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