Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils
Development of an aqueous-space mixing assay for fusion of granules and plasma membranes from human neutrophils
复制标题
DOI：
10.1042/bj3140469
复制
发表时间：
1996-03-01
影响因子：
4.1
通讯作者：
Transue, A
中科院分区：
文献类型：
--
作者：
Blackwood, RA;Smolen, JE;Transue, A
已经开发了几种模型来研究中性粒细胞脱颗粒。在最基本的水平上，磷脂微囊已经被用来研究膜融合过程中发生的脂质相互作用。用于测量磷脂囊泡融合的两种主要分析形式要么基于标记的磷脂在两个融合伙伴的膜内的稀释，要么基于囊泡的水含量的混合。尽管这两种方法都存在问题，但后者被认为更准确，更能代表真正的融合。以8-氨基-1，3，6-三磺酸(ANTS)为荧光标记物，利用对二甲基溴双吡啶(DPX)的猝灭特性，建立了一种简单的水-空间混合分析方法，可用于任何密封的囊泡。我们将我们的新方法与使用磷脂酸(PA)和磷脂酰乙醇胺(PE)组成的脂质体的更传统的方法进行了比较，在钙依赖的融合方面获得了类似的结果。我们将我们的研究扩展到中性粒细胞质膜囊泡以及天青和特定颗粒与PA/PE(1：3)脂质体的融合。在低[Ca~(2+)]至500 mM时，特异粒和质膜囊泡均与PA/PE脂质体融合，而在[Ca~(2+)]高达12 mM时，天青颗粒未见融合。这些钙离子诱导融合能力的差异可能与观察到的整个细胞在分泌方面的差异有关。
Several models have been developed to study neutrophil degranulation. At the most basic level, phospholipid vesicles have been used to investigate the lipid interactions occurring during membrane fusion. The two major forms of assays used to measure phospholipid vesicle fusion are based either on the dilution of tagged phospholipids within the membrane of the two fusing partners or the mixing of the aqueous contents of the vesicles. Although problems exist with both methods, the latter is considered to be more accurate and representative of true fusion. Using 8-aminonaphthalene-1,3,6-trisulphonic acid (ANTS) as a fluorescent marker, we have taken advantage of the quenching properties of p-xylenebispyridinium bromide ('DPX') to develop a simple aqueous-space mixing assay that can be used with any sealed vesicle. We compared our new assay with more conventional assays using liposomes composed of phosphatidic acid (PA) and phosphatidylethanolamine (PE), obtaining comparable results with respect to Ca2+-dependent fusion. We extended our studies to measure the fusion of neutrophil plasma-membrane vesicles as well as azurophil and specific granules with PA/PE (1:3) liposomes. Both specific granules and plasma-membrane vesicles fused with PA/PE liposomes at [Ca2+] as low as 500 mu M, while azurophil granules showed no fusion at [Ca2+] as high as 12 mM. These differences in the ability of Ca2+ to induce fusion may be related to differences observed in whole cells with respect to secretion.