Calcium-activated thiol-proteinase activity in the fusion of rat erythrocytes induced by benzyl alcohol.
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.
D000818:Animals; D001592:Benzyl Alcohols / Q000494:pharmacology*; D001593:Benzyl Compounds / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology*; D002459:Cell Fusion / Q000187:drug effects; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000097:blood*; D004910:Erythrocyte Membrane / Q000378:metabolism / Q000648:ultrastructure; D004912:Erythrocytes / Q000187:drug effects / Q000201:enzymology*; D066298:In Vitro Techniques; D008565:Membrane Proteins / Q000097:blood; D008854:Microscopy, Electron; D008858:Microscopy, Phase-Contrast; D051381:Rats; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D001592:Benzyl Alcohols; D001593:Benzyl Compounds; D008565:Membrane Proteins; D013439:Sulfhydryl Reagents; D010450:Endopeptidases; D003546:Cysteine Endopeptidases; D002118:Calcium