Characteristics of a sulphydryl group essential for sodium exchange diffusion in beef erythrocytes

Abstract

1. In the beef red blood cell, the component of the Na efflux which is insensitive to ouabain but depends on the presence of external Na, is not affected by furosemide but is reduced by several agents: ethacrynic acid, dinitrofluorobenzene, p-chloromercuribenzene (PCMB), N-ethylmaleimide and p-chloromercuribenzene sulphonate (PCMBS). Some of these agents increased a parallel passive permeability which could mask the reduction of efflux.2. N-ethylmaleimide and PCMBS, which in our experimental conditions (initial concentration 5 x 10(-4)M and 5 x 10(-6)M respectively, haematocrit 7.7%) do not increase the leak, inhibit Na influx and efflux markedly and equally. This provides further evidence of the existence of a typical ouabain-insensitive Na exchange diffusion in beef red blood cell.3. Inhibition of the exchange diffusion mechanism by N-ethylmaleimide or PCMBS is not total and their inhibitory effects are slightly additive. Various arguments suggest that their effects on exchange diffusion can be attributed to a reaction with sulphydryl groups.4. These sulphydryl groups are rapidly titrable by a poorly penetrating agent such as PCMBS, and the inhibitory effect is rapidly reversible. Thus, it is assumed that the sulphydryl groups containing proteins are superficially located on the outer border of the membrane.5. After inhibition, there is no change in half saturation constant for the complexing reaction for transfer, suggesting that the inhibited sites are no longer functioning but that the uninhibited sites are in every way normal.6. N-ethylmaleimide and PCMBS act similarly in sheep red blood cells.7. PCMBS does not affect sodium movement in human erythrocytes, but N-ethylmaleimide inhibits markedly the ouabain-insensitive Na efflux.