Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol

Abstract

Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. Cell fusion was inhibited by EGTA [ethylene-glycol bis[2-aminoethyl ether]-N,N''-tetraacetic acid], N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulfonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. Gel electrophoresis of membrane proteins from ghosts of the erythrocytes treated with benzyl alcohol showed that a high MW polymer was present; this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. In the treated cells the proteins corresponding to bands 2 and 3 in human erythrocytes were decreased, and a polypeptide with a slightly greater mobility than band 3 was produced. These changes were inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine and Tos-Lys-CH2Cl, but not by phenylmethanesulphonyl fluoride, Tos-Arg-OMe or histamine. The intramembranous particles of the P-fracture face of cells treated with benzyl alcohol to induce fusion were decreased in number and were susceptible to cold-induced aggregation; both of these phenomena were markedly inhibited by EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. A Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be an essential feature of membrane fusion in this system. This proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.