DIRECT CARDIOLIPIN ASSAY IN YEAST USING THE RED FLUORESCENCE EMISSION OF 10-N-NONYL ACRIDINE-ORANGE
DIRECT CARDIOLIPIN ASSAY IN YEAST USING THE RED FLUORESCENCE EMISSION OF 10-N-NONYL ACRIDINE-ORANGE
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DOI：
10.1111/j.1432-1033.1995.0113o.x
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发表时间：
1995-02-15
期刊：
影响因子：
--
通讯作者：
JULIEN, R
中科院分区：
文献类型：
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作者：
GALLET, PF;MAFTAH, A;JULIEN, R
染料10-N-壬基-3，6-双（二甲氨基）吖啶（10-N-壬基吖啶橙子）最近被鉴定为心磷脂的特异性探针（K-a = 2 × 10（6）M（-1））。它还以较低的亲和力（Ka = 7 × 10（4）M（-1））与其它酸性磷脂相互作用[Petit，J.M.，Maftah，A.，Ratinaud，M. H.和Julien，R.（1992）Eur. J. Biochem.209，267-273]。为了减少与一元酸磷脂结合的干扰，我们用荧光法定量心磷脂，该方法是基于在与二元酸磷脂接触时形成的染料二聚体的红色荧光，因此我们证明：（a）在酵母中，无论细胞代谢如何，二聚体都是染料的靶标;（B）膜或蛋白质组织和脂肪酸不饱和不显著改变10-N-壬基吖啶橙子的结合。在心磷脂的量和染料发射的红色荧光之间建立了线性关系。低红色荧光也观察到囊泡含有磷脂酰丝氨酸和磷脂酰肌醇。然而，在相同的酸性磷脂浓度下，使用含心磷脂的囊泡的荧光要高得多（是用含磷脂酰丝氨酸的囊泡观察到的荧光的五倍）。因此，将10-N-壬基吖啶橙子应用于酵母中心磷脂的定量。这种新方法显示，在高葡萄糖浓度下生长的细胞含有2.2+/-0.3 nmol心磷脂/10（6）个细胞，而在乳酸盐下，它们含有约两倍于此的量（3.9+/-0.3 nmol心磷脂）。
The dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) has been recently identified as a specific probe for cardiolipin (K-a = 2X10(6)M(-1)). It also interacts, at lower affinity (K-a = 7X10(4)M(-1)), with other acidic phospholipids [Petit, J. M., Maftah, A., Ratinaud, M. H. and Julien, R. (1992) Eur. J. Biochem. 209, 267-273]. In order to reduce the interference corresponding to monoacidic phospholipid binding, we have quantified cardiolipin by using a fluorimetric method based on the red fluorescence of the dye dimers formed at the diacidic phospholipid contact.Hence we have demonstrated that: (a) in yeast, the mitochondrion is the target of the dye whatever the cell metabolism; (b) membrane or protein organization and fatty acid unsaturation do not significantly modify the binding of 10-N-nonyl acridine orange.Using thin-walled vesicles, a linear relationship was established between the amount of cardiolipin and the red fluorescence emitted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at the same acidic phospholipid concentration, the fluorescence was much higher using cardiolipin-containing vesicles (fivefold that observed with phosphatidylserine-containing vesicles). Thus, 10-N-nonyl acridine orange was applied to cardiolipin quantification in yeast. This new method revealed that cells growing with a high glucose concentration contained 2.2+/-0.3 nmol cardiolipin/10(6) cells, whereas with lactate they contained about twice this amount (3.9+/-0.3 nmol cardiolipin).