WATER TRANSPORT IN HUMAN RED-CELLS - EFFECTS OF NONINHIBITORY SULFHYDRYL-REAGENTS ON MEMBRANE-PROTEINS AND WATER EXCHANGE

The water diffusional permeability of human red blood cells and alterations of their membrane proteins following exposure to various sulfhydryl group (SH) reagents have been studied using a nuclear magnetic resonance (NMR) technique and polyacrylamide gel electrophoresis. Exposure of red blood cells to millimolar concentrations of N-ethylmaleimide (NEM) or 5,5'-dithio bis(2-nitrobenzoic acid) (DTNB) alone does not affect water diffusion. In contrast, when DTNB treatment follows a preincubation of the cells with NEM, a small (18%) but significant inhibition of water permeability occurs. Incubation of red blood cells with 2 mM NEM for 1 hr at 25-degrees-C alone or followed by subsequent incubation with 1 mM p-chloromercuribenzene sulfonate (PCMBS), as well as incubation with DTNB does not affect the electrophoretic gel pattern of the membrane proteins. In contrast, incubation of the cells with 12 mM NEM for 1 hr at 37-degrees-C markedly affects the gel pattern. A decrease occurs in the amounts of protein of relatively high M(r), migrating as bands 1-3, and this is accompanied by an increase in protein material with M(r) lower than 70 kD, including the appearance of additional bands in the region 4.5. No further alterations could be noticed after subsequent incubation with DTNB or PCMBS. The significance of the findings is discussed in relation to the molecular mechanisms of water permeation in red blood cells and some apparently conflicting observations on the membrane proteins involved in this process.