10N‐Nonyl acridine orange interacts with cardiolipin and allows the quantification of this phospholipid in isolated mitochondria

Abstract

The acridine orange derivative, l0N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/ mol cardiolipin and 1 mol dye/mol phosphatidylscrine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 1 O6 M ~ for cardiolipin-I ON-nonyl-acridine-orange association and only 7 x lo4 M ~ ' for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction betwcen the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.The 10N-nonyl acridine orange (NAO), which is specifically incorporated into the inner mitochondrial membrane [l], plays a prominent role in the study of mitochondria in whole cells [2, 31. It enables monitoring of mitochondria in different situations, such as the cell cycle [4] and cell ageing [5] and also discrimination between different subpopulations of a heterogenous cell population, according to thcir mitochondrial contents [6, 71. Nevertheless, up to now the membrane molccular species which are specifically recognized by NAO, have not been determined.The large number of inner-mitochondrial-membrane enzymes implicated in oxidative phosphorylation, differing in their conformation and biological properties require a similar lipid environment for their activity. Cardiolipin, one of the three major phospholipids present in the inner membrane [8 -121, has been reported to be essential for the activity of the ADP/ATP carrier protein [I 31, for the phosphate transport protein [14] and for various other enzyme complexes [15,16]. This phospholipid has also been reported to be associated with the F1-FO ATPase [17]. Consequently, the NAO inhibition of the inner-mitochondrial-membrane enzymes [ 181 may be due to the interaction between the positively charged dye and cardiolipin, the main acidic phospholipid present in the inner mitochondrial membrane.The purpose ofthis work was to establish, with reference to different model membranes, that lON-nonyl acridine orange intcracts with acidic phospholipids and, more particularly, with cardiolipin. The absorbance spectra of NAO incubated with liposomes and measurements of the degree of saturation allowed us to determine the specificity and the stoichiometry of NAO ...

Photodynamic therapy: A mitochondrial inducer of apoptosis

“…This indicates that the organelle structure was preserved, since NAO labels mitochondria independent of DC m 24 via an interaction with cardiolipin. 25 Using Western blots, we demonstrated that cytochrome c was released into the cytosol immediately after the irradiation of porphycene-sensitized cells at 108C ( Figure 2). The control (lane A) provides an estimate of the extent of mitochondrial contamination and/or base-line mitochondrial damage in the cell extracts used in this assay.…”

Enzymatic oxidation products of spermine induce greater cytotoxic effects on human multidrug‐resistant colon carcinoma cells (LoVo) than on their wild‐type counterparts

“…29 LoVo cells were detached with EDTA/trypsin, then washed, centrifuged and resuspended in complete F12 medium. Cells (5 ϫ 10 5 /ml) were labeled with 20 nM NAO for 30 min at 37°C and after washing with cold PBS, samples were investigated by flow cytometry.…”

The c-Myc target gene PRDX3 is required for mitochondrial homeostasis and neoplastic transformation

“…However, R1a-myc-PRDX3AS cells show a minimal increase in reactive oxygen species. Analysis of mitochondrial mass with 10-N-nonyl-acridine orange (NAO) (27) reveals that MCF7͞ADR-PRDX3AS cells show decreased mitochondrial mass, whereas R1a-myc-PRDX3AS cells also show a small percentage of cells with reduced mitochondrial mass. Reduction of PRDX3 in both cell lines results in a decrease in mitochondrial membrane potential, indicated by the reduced uptake of 3,3Ј-dihexyloxacarbocyanine iodide (DiOC 6 ), as shown in Fig.…”