Leto T L, Roseman M A, Holloway P W
Biochemistry. 1980 Apr 29;19(9):1911-6. doi: 10.1021/bi00550a028.
The intervesicle exchange of cytochrome b5 has been studied by fluorescence quenching. The binding of cytochrome b5 to 1,2-bis(9.10-dibromostearoyl)-sn-glycerol-3-phosphorylcholine vesicles results in a quenching of cytochrome b5 fluorescence whereas the fluorescence is enhanced upon binding to 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine vesicles. This difference in cytochrome b5 fluorescence upon binding was used to study the kinetics of cytochrome b5 intervesicle exchange between the "quenching" and "enhancing" vesicles. Separation of the two cytochrome b5-vesicle complexes by density gradient centrifugation provided direct evidence for cytochrome b5 intervesicle exchange. Both the fluorescence assay and the density gradient assay yield the same value for the extent of cytochrome b5 exchange, obtained after equilibration, between the two types of vesicles. Both experiments also indicate that cytochrome b5 binds in a reversible fashion and has an equal affinity for the two types of vesicles. The kinetics of the exchange process are consistent with a mechanism involving the transfer of cytochrome b5 through the aqueous phase and rule out a mechanism involving vesicle collision.
已通过荧光猝灭研究了细胞色素b5在囊泡间的交换。细胞色素b5与1,2-双(9,10-二溴硬脂酰基)-sn-甘油-3-磷酸胆碱囊泡结合会导致细胞色素b5荧光猝灭，而与1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸胆碱囊泡结合时荧光增强。利用细胞色素b5结合时荧光的这种差异来研究细胞色素b5在“猝灭”囊泡和“增强”囊泡之间进行囊泡间交换的动力学。通过密度梯度离心分离两种细胞色素b5-囊泡复合物为细胞色素b5的囊泡间交换提供了直接证据。荧光测定法和密度梯度测定法对于平衡后两种类型囊泡之间细胞色素b5的交换程度得出了相同的值。两个实验还表明细胞色素b5以可逆方式结合，并且对两种类型的囊泡具有相同的亲和力。交换过程的动力学与涉及细胞色素b5通过水相转移的机制一致，排除了涉及囊泡碰撞的机制。
