Vol. 111, No. 3, 1983 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS March 29, 1983 Pages 970-979 EFFECT OF HEMOLYSATE ON CALCIUM INHIBITION OF THE (Na+ + K+)-ATPase OF HUMAN RED BLOOD CELLS Douglas R. Yingst and Michael J. Marcovitz Department of Physiology, Wayne State University School of Medicine, 'Detroit, Michigan 48201 Received February 14, 1983 The sensitivity of the (Nat + K+)-ATPase in human red cell membranes to inhibition by Ca*+ is markedly increased by the addition of diluted cytoplasm from hemolyzed human red blood cells. The concentration of Ca*+ causing 50% inhibition of the (Nat + Kt)-ATPase is shifted from greater Cazt in the absence of hemolysate to less than 10 uM free Ca j?a;hz; uM free hemolysate diluted 1:6U compared to in vivo concentrations is added to the -- assay mixture. Boiling the hemolysate destroys its ability to increase the sensitivity of the (Na+ + K+)-ATPase to Cazt. Proteins extracted from the membrane in the presence of EDTA and concentrated on an Amicon PM 30 membrane increased the sensitivity of the (Nat f Kt)-ATPase to Ca*+ in a dose-dependent fashion, causing over 80% inhibition of the (Nat + Kt)-ATPase at 10 $I free Ca*+ at the highest concentration of the extract tested. The active factor in this membrane extract is Ca*+-dependent, in the absence of Ca*+. because it had no effect on the (Nat + Kt)-ATPase Trypsin digestion prior to the assay destroyed the ability of this rotein extract to increase the sensitivity of the (Nat + Kf)-ATPase to Ca 8t . It has been known for some time that Ca*' inhibits the (Nat + K')-pump (1) and the (Nat t K+)-ATPase (2) of human red blood cells, as it does the (Nat t Kt)-ATPase from other cells (3,4). The concentration of intracellular free Ca*' causing 50% inhibition of the (Nat + K')-pump in human red cells has been estimated to be approximately 100 IJM (5) which is reasonably close to the concentration of Ca*+ causing 50% inhibition of the (Nat + Kt)-ATPase in white unsealed membranes from human red cells (2). More recently it was reported that the (Nat + K+)-pump of resealed human red cell ghosts Abbreviations used: Hepes, N-2-hydroxyethylpiperazine-N'-Z-ethanesulfonic acid; EGTA, ethyleneglycol-bis(B-aminoethyl ether)N,N'-tetraacetic acid; and EDTA, ethylenediaminetetraacetic acid. 0006291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. 970