PREPARATION AND CHARACTERIZATION OF PLASMA-MEMBRANE VESICLES FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES
PREPARATION AND CHARACTERIZATION OF PLASMA-MEMBRANE VESICLES FROM HUMAN POLYMORPHONUCLEAR LEUKOCYTES
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DOI：
10.1002/jcp.1041410323
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发表时间：
1989-12-01
影响因子：
5.6
通讯作者：
SIMONS, ER
中科院分区：
文献类型：
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作者：
DELBUONO, BJ;LUSCINSKAS, FW;SIMONS, ER
制备中性粒细胞质膜的模型将是有利的，以检查质膜在人中性粒细胞中跨膜信号转导中的作用，并剖析刺激后细胞表面的配体-受体相互作用和结构变化。许多研究者已经通过匀浆、超声或离心技术制备了中性粒细胞膜囊泡，这些技术可能导致大量表面膜材料的损失、溶酶体的破坏导致膜蛋白的蛋白水解以及质膜部分被内膜污染。这些局限性已被克服，在本研究中采用的方法在本实验室以前开发的修改。将人中性粒细胞悬浮在模拟细胞质离子和渗透条件的缓冲液中，并通过氮气空化破坏。将所得的空化液中未破碎的细胞和细胞核清除，然后通过不连续的等渗/等渗Percoll梯度离心，分离出四种组分：α（完整的嗜天青颗粒）、β（完整的特异性颗粒）、γ（膜囊泡）和δ（胞质溶胶）。γ组分高度富集碱性磷酸酶，这是质膜的标志物。此外，该级分含有完整细胞或未分级空化液中发现的溶酶体（由溶菌酶活性指示）和细胞核（由DNA含量指示）的<5%。此外，通过葡萄糖6-磷酸酶、半乳糖基转移酶、单胺氧化酶和Mo 1（CD 11b/CD 18; Mac-1）测定，γ组分中的内质网、高尔基体、线粒体和溶酶体膜水平分别<10%。最后，如哇巴因敏感性（Na+，K+）ATP酶活性测定所示，75%的膜囊泡被密封，如通过暴露囊泡表面上的伴刀豆球蛋白A（ConA）受体和唾液酸残基所确定的，55%的膜囊泡被定向为正面向外。这些异质性制备物可以通过选择性粘附于ConA包被的平板并随后通过用α-甲基甘露糖苷冲洗平板表面来分离，从而富集右侧外囊泡。该富集方案不影响囊泡的完整性，并产生其中>85%的囊泡朝向右侧的群体。因此，该程序允许制备密封的、正面朝外的膜囊泡，其可用作各种功能研究中中性粒细胞质膜的有效实验模型。
It would be advantageous to prepare models of the neutrophil plasma membrane in order to examine the role of the plasma membrane in transmembrane signal transduction in the human neutrophil and to dissect ligand‐receptor interactions and structural changes in the cell surface upon stimulation. A number of investigators have prepared neutrophil membrane vesicles by homogenization, sonication, or centrifugation–techniques that can result in the loss of substantial amounts of surface membrane material, disruption of lysosomes causing proteolysis of membrane proteins, and contamination of the plasma membrane fraction by internal membranes. These limitations have been overcome in the present studies by employing a modification of the method previously developed in this laboratory. Human neutrophils were suspended in a buffer simulating cytoplasmic ionic and osmotic conditions and disrupted by nitrogen cavitation. The resultant cavitate was freed of undisrupted cells and nuclei and then centrifuged through discontinuous isotonic/isoosmotic Percoll gradients, which resolved four fractions: α (intact azurophilic granules), β (intact specific granules), γ (membrane vesicles), and δ (cytosol). The γ fraction was highly enriched in alkaline phosphatase, a marker of the plasma membrane. In addition, this fraction contained <5% of the amounts of lysosomes (indicated by lysozyme activity) and nuclei (indicated by DNA content) found in intact cells or in unfractionated cavitate. Furthermore, the γ fraction contained <10% of the levels of endoplasmic reticulum, Golgi, mitochondrial, and lysosomal membranes in cells or cavitates, as determined by assays for glucose 6‐phosphatase, galactosyl transferase, monoamine oxidase, and Mo1 (CD11b/CD18; Mac‐1), respectively. Finally, 75% of the membrane vesicles were sealed, as indicated by assay of ouabain‐sensitive (Na+, K+) ATPase activity, and 55% were oriented right‐side‐out, as determined by exposure of concanavalin A (ConA) receptors and sialic acid residues on the surfaces of the vesicles. These heterogeneous preparations could be enriched for right‐side‐out vesicles by their selective adherence to ConA‐coated plates and subsequent detachment by rinsing the surfaces of the plates with α‐methylman‐noside. This enrichment protocol did not affect the integrity of the vesicles and resulted in populations in which >85% of the vesicles were oriented right‐sideout. This procedure thus permits the preparation of sealed, right‐side‐out membrane vesicles that may be used as valid experimental models of the neutrophil plasma membrane in a variety of functional studies.