Taylor K M, Roseman M A
Department of Biochemistry, Uniformed Services University of the Health Sciences, Bethesda, MD 20814-4799, USA.
Biochim Biophys Acta. 1996 Jan 12;1278(1):35-40. doi: 10.1016/0005-2736(95)00195-6.
When cytochrome b5 is added to large unilamellar vesicles (LUVs) of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), it binds predominantly in a 'loose,' or transferable form. Prolonged incubation of 30 degrees C leads to insertion in the physiological 'tight,' nontransferable form, with a halftime for the loose --> tight conversion of approx. 9 days. In this study, the effect of cholesterol on the rate of tight insertion was determined. Tight binding was assayed by depleting the LUVs of loose cytochrome b5 with an excess of SUV acceptors and then separating the liposome populations by gel-filtration or velocity sedimentation. Incorporation of cholesterol into the LUVs was found to markedly increase the rate of tight insertion, even though cholesterol decreases the equilibrium binding constant and saturation level of protein binding. The effect is not a continuously increasing function of cholesterol content, but attains a maximum at 20-25% mol%, where the rate enhancement is approx. 10-fold over baseline. At higher cholesterol levels, the rate decreases, returning to baseline at 40 mol% cholesterol. These observations are highly unusual in that cholesterol generally decreases the membrane binding affinity and the permeability of solutes, and does so as a monotonic function of cholesterol concentration (above the liquid-crystalline phase transition of the phospholipids). It is suggested that tight insertion is enhanced by lipid-protein packing mismatches and by bilayer fluidity; the former increases monotonically with increasing cholesterol whereas the latter decreases monotonically. At 20-25 mol% cholesterol the optimum balance of these physical properties is obtained for tight insertion.
当细胞色素b5添加到1-棕榈酰-2-油酰磷脂酰胆碱（POPC）的大单层囊泡（LUVs）中时，它主要以“松散”或可转移的形式结合。在30摄氏度下长时间孵育会导致其以生理上的“紧密”、不可转移的形式插入，从松散形式转变为紧密形式的半衰期约为9天。在本研究中，测定了胆固醇对紧密插入速率的影响。通过用过量的小单层囊泡（SUV）受体耗尽LUVs中松散的细胞色素b5，然后通过凝胶过滤或速度沉降分离脂质体群体来测定紧密结合。发现将胆固醇掺入LUVs中会显著提高紧密插入的速率，尽管胆固醇会降低蛋白质结合的平衡结合常数和饱和水平。这种影响不是胆固醇含量的连续增加函数，而是在20 - 25%摩尔百分比时达到最大值，此时速率增强约为基线的10倍。在胆固醇水平较高时，速率下降，在胆固醇含量为40摩尔百分比时回到基线。这些观察结果非常不寻常，因为胆固醇通常会降低膜结合亲和力和溶质的通透性，并且是磷脂液晶相转变温度以上胆固醇浓度的单调函数。有人认为，紧密插入通过脂质 - 蛋白质堆积不匹配和双层流动性得到增强；前者随胆固醇增加而单调增加，而后者单调减少。在胆固醇含量为20 - 25摩尔百分比时，获得了紧密插入这些物理性质的最佳平衡。
