Thiol-dependent passive K/Cl transport in sheep red cells: III. Differential reactivity of membrane SH groups with N-ethylmaleimide and iodoacetamide.

Abstract

Treatment with N-ethylmaleimide (NEM) is known to stimulate ouabain-insensitive, Cl- -dependent K+ transport in low K+ (LK) but not in high K+ (HK) sheep red cells (Lauf, P.K., and Theg, B.E., 1980, Biophys. Biochem. Res. Commun. 92:1422-1428). The dependence of this effect on the pH of pretreatment with NEM and/or iodoacetamide (IAA) was studied. Maximum stimulation of Cl- -dependent K+ transport in LK red cells was produced by prior treatment with 1-5 mM NEM at pH 6 at which only about 30-40% of the 10(7) SH groups present per membrane reacted. At pH 6 no NEM effect was seen on Nap K+ fluxes in HK red cells. Treatment with NEM below pH 6 enhanced Cl- -independent K+ transport in both LK and HK red cells. At higher pH values or higher concentrations the NEM-stimulation of K+ transport was reduced and absent at pH 8.7. Exposure of LK cells to 5 mM IAA prior to NEM abolished the stimulatory effect of NEM on K+ transport. Hence at least two different chemical groups were reacting with NEM, and more alkaline SH or NH2 groups whose reaction with NEM leads to an inhibition of the NEM effect brought about at pH 6.