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

Abstract

1. Rat erythrocytes were fused by incubation with benzyl alcohol and Ca2+. 2. Cell fusion was inhibited by EGTA, N-ethylmaleimide, tetrathionate, iodoacetamide, cystamine, Tos-Lys-CH2Cl, and to a lesser extent by Tos-Phe-CH2Cl. Phenylmethanesulphonyl fluoride, Tos-Arg-OMe and histamine did not inhibit cell fusion. 3. Gel electrophoresis of membrane proteins from "ghosts" of the erythrocytes treated with benzyl alcohol showed that a high-molecular-weight polymer was present: this was consistent with the entry into the cells of Ca2+ and the activation of a transglutaminase enzyme. 4. 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. 5. 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. 6. The intramembraneous 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 to EGTA, and partially inhibited by Tos-Lys-CH2Cl and N-ethylmaleimide. 7. These several observations indicate that a Ca2+-activated thiol-proteinase, which acts to degrade membrane proteins and to give freedom of lateral movement to intramembranous particles, may be essential feature of membrane fusion in this system. 8. It is suggested that this proteinase may act to degrade spectrin-binding proteins that attach band-3 protein to the erythrocyte cytoskeleton.

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

“…Calcium seems to be a requirement for most types of fusion; it has been suggested that the function of fusion-promoting agents is to introduce calcium into the cell (33). The actual role of protease activity in the overall fusion process has not been clarified (31,33,34). We show here that the introduction of calcium per se and the activation of protease activity do not suffice for the induction of fusion.…”

“…The protease(s) necessary for fusion could be activated directly or indirectly (31) (i.e., via another enzyme) by calcium. Calcium seems to be a requirement for most types of fusion; it has been suggested that the function of fusion-promoting agents is to introduce calcium into the cell (33). The actual role of protease activity in the overall fusion process has not been clarified (31,33,34).…”

“…Protease activity may be necessary for a variety of natural and experimental membrane fusion events (1,(31)(32)(33)(34). Ca2+-activated proteases are found in a variety of cells (35)(36)(37), but the possible connection of any of these to membrane fusion has not been previously established.…”

Do hydrophobic sequences cleaved from cellular polypeptides induce membrane fusion reactions in vivo?

“…Unlike the fusion of human erythrocytes by chlorpromazine and oleoylglycerol, the fusion of rat red blood cells by benzyl alcohol and the associated degradation of their membrane proteins is prevented by agents that chelate Ca2+ and by iVethylmaleimide [20]. This indicates that the Cat+-activated cysteine proteinase is more important in these cells, and it is interesting that the content of calpastatin, an endogenous inhibitor of the Ca'+-activated, cysteine proteinase known as calpain I is extremely small in rat erythrocytes, whiIe in human erythrocytes there is much more inhibitor than enzyme [21].…”

An osmotic model for the fusion of biological membranes

“…Proteinases are important in chemically induced cell fusion , and the fusion of erythrocytes by some chemical fusogens involves the proteolysis of ankyrin by a serine proteinase [23] and the proteolysis of band 3 by a calcium-activated proteinase [23,24]. Inhibitors of these proteolytic activities inhibit fusion.…”