Lang R D, Wickenden C, Wynne J, Lucy J A
Biochem J. 1984 Mar 1;218(2):295-305. doi: 10.1042/bj2180295.
Human erythrocytes were fused by incubation with 0.5-2 mM-chlorpromazine hydrochloride at pH 6.8-7.6. Fusogenic preparations of chlorpromazine were cloudy suspensions of microdroplets, and below pH 6.8 chlorpromazine gave clear solutions that were inactive. Unlike control cells, the lateral mobility of the intramembranous particles of the PF-fracture face of chlorpromazine-treated cells was relatively unrestricted, since the particles were partly clustered at 37 degrees C and they exhibited extensive cold-induced clustering. Ca2+ stimulated fusion, but fusion was only very weakly inhibited by EGTA (10 mM) and by N-ethylmaleimide (50 mM); pretreatment of the cells with Tos-Lys-CH2Cl (7-amino-1-chloro-3-L-tosylamidoheptan-2-one) (7.5 mM) markedly inhibited fusion. Changes in the membrane proteins of erythrocytes fused by chlorpromazine, before and after treatment with chymotrypsin to remove band 3 protein, were investigated. The several observations made indicate that the Ca2+-insensitive component of fusion is associated with degradation of ankyrin (band 2.1 protein) to band 2.3-2.6 proteins and to smaller polypeptides by a serine proteinase that is inhibited by Tos-Lys-CH2Cl, and that the component of fusion inhibited by EGTA and N-ethylmaleimide is associated with degradation of band 3 protein to band 4.5 protein by a Ca2+-activated cysteine proteinase. Proteolysis of ankyrin appeared to be sufficient to permit the chlorpromazine-induced fusion of human erythrocytes, but fusion occurred more rapidly when band 3 protein was also degraded in the presence of Ca2+. Since other cells have structures comparable with the spectrin-actin skeleton of the erythrocyte membrane, the observations reported may be relevant to the initiation of naturally occurring fusion reactions in biomembranes. It is also suggested that, should polypeptides with fusogenic properties be produced from integral and skeletal membrane proteins by endogenous proteolysis, their formation would provide a general mechanism for the fusion of lipid bilayers in biomembrane fusion reactions.
将人红细胞与0.5 - 2 mM盐酸氯丙嗪在pH 6.8 - 7.6条件下孵育可使其发生融合。氯丙嗪的促融合制剂为微滴的浑浊悬浮液，在pH 6.8以下，氯丙嗪形成的是无活性的澄清溶液。与对照细胞不同，经氯丙嗪处理的细胞的PF - 断裂面膜内颗粒的侧向流动性相对不受限制，因为这些颗粒在37℃时部分聚集，并且它们表现出广泛的冷诱导聚集。Ca²⁺刺激融合，但EGTA（10 mM）和N - 乙基马来酰亚胺（50 mM）对融合的抑制作用非常微弱；用Tos - Lys - CH₂Cl（7 - 氨基 - 1 - 氯 - 3 - L - 甲苯磺酰氨基庚烷 - 2 - 酮）（7.5 mM）对细胞进行预处理可显著抑制融合。研究了用氯丙嗪融合的红细胞在用胰凝乳蛋白酶处理以去除带3蛋白前后膜蛋白的变化。所做的几项观察表明，融合的Ca²⁺不敏感成分与锚蛋白（带2.1蛋白）被一种被Tos - Lys - CH₂Cl抑制的丝氨酸蛋白酶降解为带2.3 - 2.6蛋白及更小的多肽有关，而被EGTA和N - 乙基马来酰亚胺抑制的融合成分与带3蛋白被一种Ca²⁺激活的半胱氨酸蛋白酶降解为带4.5蛋白有关。锚蛋白的蛋白水解似乎足以允许氯丙嗪诱导的人红细胞融合，但当在Ca²⁺存在下带3蛋白也被降解时，融合发生得更快。由于其他细胞具有与红细胞膜血影蛋白 - 肌动蛋白骨架相当的结构，所报道的观察结果可能与生物膜中自然发生的融合反应的起始有关。还提出，如果通过内源性蛋白水解从整合膜蛋白和骨架膜蛋白产生具有促融合特性的多肽，它们的形成将为生物膜融合反应中脂质双层的融合提供一种普遍机制。
