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...