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The cellular free magnesium concentration of ionophore A23187 permeabilized high potassium sheep erythrocytes was measured by 31P nuclear magnetic resonance spectroscopy, and the total cellular magnesium concentration was determined by atomic absorption spectroscopy. The free versus total cellular magnesium concentrations yield a linear relationship on a log-log scale in the concentration range from 0.3 to 1.92 mmol Mg/liter cells. Thus, free intracellular magnesium concentrations can be calculated from atomic absorption data. The method permits the estimation of physiologically or experimentally induced variations of intracellular free magnesium concentrations between 7 and 405 microM magnesium in cell water. This range encompasses the free magnesium concentration of 335 +/- 60 microM in cell water determined for untreated erythrocytes.

In red cells of several species, the sulfhydryl reagent N-ethylmaleimide activates a Cl- -dependent, ouabain-resistant K+ transport pathway. Here we report our attempts to demonstrate ouabain-resistant Cl- -dependent K+ fluxes stimulated by N-ethylmaleimide in resealed human red cell ghosts using Rb+ as a K+ analogue. In contrast to intact cells, the rate constants of the base level Rb+ efflux in ghosts were similar in NaNO3 and NaCl (okRb = 0.535 +/- 0.079 h-1 and 0.534 +/- 0.085 h-1, respectively), while 1 mM N-ethylmaleimide stimulated Rb+ efflux strongly in NaNO3 (okRb = 14.26 +/- 1.32 h-1) and moderately in NaCl (okRb = 2.73 +/- 0.54 h-1). This effect was dependent on the presence of internal ATP. Stimulation of Rb+ efflux was observed in the presence of greater than or equal to 0.2 mM N-ethylmaleimide and increased at pH values approaching 8.0, consistent with titration of SH groups. N-Ethylmaleimide-stimulated Rb+ efflux was approx. 50% inhibited by 100 microM quinine sulfate whereas 1 microM bumetanide had no effect. In NaCl the N-ethylmaleimide-stimulated efflux saturated with initial internal ghost Rb+ concentration, but rates increased linearly in NaNO3. Replacement of external Na+ with glucamine or choline decreased the N-ethylmaleimide-stimulated Rb+ efflux, suggesting a role for external Na+. N-Ethylmaleimide-stimulated Rb+ efflux was greater in buffers with lipophilic anions such as SCN- or NO3- than in solutions with Cl- or acetate. However, the cation selectivity of the pathway studied was low, as Li+ efflux was also stimulated by N-ethylmaleimide. We conclude that the effect of N-ethylmaleimide on ouabain-resistant cation effluxes of human red cell ghosts is very different from the selective action of N-ethylmaleimide on Rb+ influxes in intact red cells.

This paper reports on several biochemical characteristics of the M antigen in membranes of high-potassium sheep red cells. 1. 1. The M antigen was solubilized by sodium deoxycholate and retained its activity following removal of detergent. 2. 2. Membrame proteins and lipids were separated by gel filtration in deoxycholate and the M antigen was shown to reside only in the protein fraction, but at reduced levels. 3. 3. Antigenic activity was enhanced by recombination with lipid, and both phospholipid and cholesterol were required for full activation. 4. 4. Treatment of whole M-positive membranes with 2-mercaptoethanol and iodoacetamide resulted in a slight decreased of the M-antigenic activity but completely inactivated the M antigen when performed in the presence of 6 M guanidine hydrochloride. The results indicate that the antigen is bound to the membrane primarily through hydrophobic forces. We suggest that portions of the antigen are relatively buried in the membrane matrix, and are inaccessible to small molecular weight reagents.