Calpain activation is essential for membrane fusion of erythrocytes in the presence of exogenous Ca2+.
The membrane mobility agent, 2-(methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes the fusion of rat, rabbit, and human erythrocytes in the presence of exogenous Ca2+. Under these conditions, the high sensitivity form of calcium-activated neutral protease (mu-calpain) in erythrocytes is activated autolytically. mu-Calpain is activated in accordance with fusion; that is, both erythrocyte fusion and autolytic activation of mu-calpain are induced in rat erythrocytes at 30 min, in rabbit erythrocytes at 150 min, and in human erythrocytes at 240 min after the addition of A2C and Ca2+. When erythrocytes are preincubated with the Ca2+ ionophore A23187, both fusion and autolytic activation start earlier. A leupeptin analogue, Cbz-Leu-Leu-Leu-aldehyde (ZLLLal), inhibits both the autolytic activation of mu-calpain and fusion induced by A2C and Ca2+. These results indicate that treatment of erythrocytes with A2C and Ca2+, results in first an influx of Ca2+ into the cells, followed by autolytic activation of mu-calpain, proteolysis of membrane proteins, exposure of fusion-sites, and, finally, fusion of erythrocytes.
Hayashi
M
M
Department of Biochemistry, Tokyo Metropolitan Institute of Gerontology, Japan.
Saito
Y
Y
Kawashima
S
S
D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002154:Calpain / Q000097:blood* / Q000302:isolation & purification; D004789:Enzyme Activation; D004910:Erythrocyte Membrane / Q000187:drug effects / Q000502:physiology* / Q000648:ultrastructure; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D007976:Leupeptins / Q000494:pharmacology; D008561:Membrane Fusion / Q000187:drug effects; D011480:Protease Inhibitors / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D013228:Stearates / Q000494:pharmacology*
D007976:Leupeptins; D011480:Protease Inhibitors; D013228:Stearates; D000001:Calcimycin; C023714:A(2)C; D002154:Calpain; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde; D002118:Calcium