Roles of lipids and proteins in the Ca2+-PO4-induced aggregation of cytoskeleton-free erythrocyte vesicle membranes.
Roles of lipids and proteins in the Ca2+-PO4-induced aggregation of cytoskeleton-free erythrocyte vesicle membranes.
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DOI：
10.1021/bi00307a028
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发表时间：
1984-06
期刊：
影响因子：
2.9
通讯作者：
K. Leonards;S. Ohki
中科院分区：
文献类型：
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作者：
K. Leonards;S. Ohki
研究了脂质和蛋白质在Ca 2 +-PO 4诱导的膜聚集中的作用。采用来自完整的人和兔红细胞（分别为HEves和REves）的无细胞因子囊泡作为模型系统。HEves和REves具有简化的膜蛋白组成[带3蛋白和糖蛋白PAS-1、-2和-3（HEves）]和正常的脂质组成。最佳实验条件的pH值，[PO 4]，和[CaCl 2]进行了测定，定量研究动态和程度的HEves和REves聚集，浊度法测量。聚集过程被认为是相当敏感的pH值和[PO 4]的微小变化和不太敏感的[CaCl 2]。膜蛋白在囊泡聚集中的作用通过选择性地修饰蛋白质来检测。通过使用由道奇鬼脂质提取物制成的超声脂质囊泡[小单层囊泡（SUV）]研究脂质的作用。用胰蛋白酶、糜蛋白酶或链霉蛋白酶进行酶处理对囊泡聚集的速率或程度（2分钟孵育期）均无影响。神经氨酸酶处理使这两个因素降低约20%。SUV与Ca ~（2+）-PO_4的聚集依赖于PO_4/脂质的比例。这些结果表明：（1）PO_4与囊泡表面有关，涉及膜脂;（2）通过酶修饰囊泡膜蛋白，消除了PO_4效应中囊泡+PO_4孵育时间分量;（3）含唾液酸分子的定性而非定量性质影响囊泡聚集;（4）除孵育时间效应外，膜蛋白既不促进也不抑制Ca ~（2+）-PO_4诱导的HEves或REves聚集。
The roles of lipids and proteins in Ca2+-PO4-induced membrane aggregation were investigated. Cytoskeleton-free vesicles derived from intact human and rabbit erythrocytes (HEves and REves, respectively) were employed as a model system. The HEves and REves have a simplified membrane protein composition [band 3 proteins and glycoproteins PAS-1, -2, and -3 (HEves)] and normal lipid composition. Optimal experimental conditions for pH, [PO4], and [CaCl2] were determined for quantitatively examining the dynamics and extent of HEves and REves aggregation, measured turbidimetrically. The aggregation process was found to be quite sensitive to small changes in pH and [PO4] and much less sensitive to [CaCl2]. The roles of membrane proteins in vesicle aggregation were examined by selectively modifying the proteins enzymatically. The roles of lipids were studied by using sonicated lipid vesicles [small unilamellar vesicles (SUVs)] made from Dodge ghost lipid extracts. Enzymatic treatment with trypsin, chymotrypsin, or Pronase had no effect on either the rates or the extent of vesicle aggregation (2-min incubation period). Neuraminidase treatment reduced both factors by approximately 20%. SUVs aggregated with Ca2+-PO4 in a way which depended on the PO4/lipid ratio. Together the results suggest the following: (1) PO4 is associated with the vesicle surface, involving the membrane lipids; (2) the vesicle + PO4 incubation time component of the PO4 effect is eliminated by enzymatically modifying the vesicle membrane proteins; (3) qualitative, rather than quantitative, properties of sialic acid containing molecules affect vesicle aggregation; and (4) with the exception of the incubation time effect, membrane proteins seem neither to promote nor to inhibit Ca2+-PO4-induced HEves or REves aggregation.