THIOL-DEPENDENT PASSIVE K-CL TRANSPORT IN SHEEP RED-BLOOD-CELLS .10. A HYDROXYLAMINE-OXIDATION INDUCED K-CL FLUX BLOCKED BY DIETHYLPYROCARBONATE

Hydroxylamine, a potent oxidizing agent used to reverse carbethoxylation of histidine by diethylpyrocarbonate, activated Cl-dependent K flux (K:Cl cotransport) of low K sheep red blood cells almost sixfold. When K:Cl cotransport was already stimulated by N-ethylmaleimide, hydroxylamine caused an additional twofold activation suggesting modification of sites different from those thiol alkylated. This conclusion was supported by the finding that hydroxylamine additively augmented also the diamide-induced K:Cl flux (Lauf, P.K. 1988. J. Membrane Biol.101:179-188) with dithiothreitol fully reversing the diamide but not the hydroxylamine effect. Stimulation of K:Cl cotransport by hydroxylamine was completely inhibited by treatment with diethylpyrocarbonate also known to prevent K:Cl cotransport stimulation by N-ethylmaleimide, both effects being independent of the order of addition. Hence, although the effect of carbethoxy modification on K:Cl flux cannot be reversed by hydroxylamine and thus excludes histidine as the target for diethylpyrocarbonate, our finding reveals an important chemical determinant of K:Cl cotransport stimulation by both hydroxylamine oxidation and thiol group alkylation. © 1989 Springer-Verlag New York Inc.