Mancheño J M, Gasset M, Lacadena J, Ramón F, Martínez del Pozo A, Oñaderra M, Gavilanes J G
Departamento de Bioquímica y Biología Molecular, Facultad de Química, Universidad Complutense, Madrid, Spain.
Biophys J. 1994 Sep;67(3):1117-25. doi: 10.1016/S0006-3495(94)80578-8.
alpha-Sarcin is a fungal cytotoxic protein that inactivates the eukaryotic ribosomes. A kinetic study of the aggregation and lipid mixing promoted by this protein on phosphatidylglycerol (PG) and phosphatidylserine (PS) vesicles has been performed. Egg yolk PG, bovine brain PS, dimyristoyl-PG (DMPG) and dimyristoyl-PS (DMPS) vesicles have been considered. The initial rates of the vesicle aggregation induced by the protein have been measured by stopped-flow 90 degrees light scattering. The formation of a vesicle dimer as the initial step of this process was deduced from the second-order dependence of the initial rates on phospholipid concentration. The highest alpha-sarcin concentration studied did not inhibit the vesicle aggregation, indicating that many protein molecules are involved in the vesicle cross-linking. These are common characteristics of the initial steps of the aggregation produced by alpha-sarcin in the four types of phospholipid vesicles considered. However, the kinetics of the scattering values revealed that more complex changes occurred in the later steps of the aggregation process of egg PG and brain PS vesicles than in those of their synthetic counterparts. alpha-Sarcin produced lipid mixing in vesicles composed of DMPG or DMPS, which was measured by fluorescence resonance energy transfer assays. A delay in the onset of the process, dependent on the protein concentration, was observed. Measurement of the rates of lipid mixing revealed that the process is first order on phospholipid concentration. Egg PG and brain PS vesicles did not show lipid mixing, although they avidly aggregated. However, alpha-sarcin was able to promote lipid mixing in heterogeneous systems composed of egg PG+DMPG or brain PS+DMPS vesicles. The dilution of the fluorescence probes was faster when these were incorporated into the bilayers made of synthetic phospholipids than were present in those made of natural phospholipids. The bilayer destabilization produced by the protein in the vesices composed of the dimyristoyl-phospholipids should be transmitted to the more stable ones made of natural phospholipids. The obtained results are interpreted in terms of lipid mixing occurring within vesicle aggregates larger than dimer.
α-肌动蛋白是一种能使真核核糖体失活的真菌细胞毒性蛋白。已对该蛋白在磷脂酰甘油（PG）和磷脂酰丝氨酸（PS）囊泡上促进的聚集和脂质混合进行了动力学研究。研究了蛋黄PG、牛脑PS、二肉豆蔻酰-PG（DMPG）和二肉豆蔻酰-PS（DMPS）囊泡。通过停流90度光散射测量了该蛋白诱导囊泡聚集的初始速率。该过程的初始步骤是形成囊泡二聚体，这是根据初始速率对磷脂浓度的二级依赖性推导出来的。所研究的最高α-肌动蛋白浓度并未抑制囊泡聚集，这表明许多蛋白质分子参与了囊泡交联。这些是α-肌动蛋白在四种类型的磷脂囊泡中产生聚集的初始步骤所共有的特征。然而，散射值的动力学表明，与合成对应物相比，卵PG和脑PS囊泡聚集过程后期发生了更复杂的变化。α-肌动蛋白在由DMPG或DMPS组成的囊泡中产生脂质混合，这通过荧光共振能量转移测定法进行测量。观察到该过程开始存在延迟，这取决于蛋白质浓度。脂质混合速率的测量表明，该过程对磷脂浓度呈一级反应。卵PG和脑PS囊泡虽然能强烈聚集，但未显示脂质混合。然而，α-肌动蛋白能够在由卵PG+DMPG或脑PS+DMPS囊泡组成的异质系统中促进脂质混合。当荧光探针掺入由合成磷脂制成的双层中时，其稀释速度比存在于由天然磷脂制成的双层中时更快。由该蛋白在由二肉豆蔻酰磷脂组成的囊泡中产生的双层不稳定应传递到由天然磷脂制成的更稳定双层中。所得结果根据大于二聚体的囊泡聚集体内发生的脂质混合进行解释。
