DIRECT CARDIOLIPIN ASSAY IN YEAST USING THE RED FLUORESCENCE EMISSIONOF 10-N-NONYL ACRIDINE-ORANGE

The dye 10-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) has been recently identified as a specific probe for cardioli pin (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, 267-27 3]. In order to reduce the interference corresponding to monoacidic ph ospholipid binding, we have quantified cardiolipin by using a fluorime tric 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 ce ll metabolism; (b) membrane or protein organization and fatty acid uns aturation do not significantly modify the binding of 10-N-nonyl acridi ne orange. Using thin-walled vesicles, a linear relationship was estab lished between the amount of cardiolipin and the red fluorescence emit ted by the dye. Low red fluorescences were also observed with vesicles containing phosphatidylserine and phosphatidylinositol. However, at t he same acidic phospholipid concentration, the fluorescence was much h igher using cardiolipin-containing vesicles (fivefold that observed wi th phosphatidylserine-containing vesicles). Thus, 10-N-nonyl acridine orange was applied to cardiolipin quantification in yeast. This new me thod revealed that cells growing with a high glucose concentration con tained 2.2+/-0.3 nmol cardiolipin/10(6) cells, whereas with lactate th ey contained about twice this amount (3.9+/-0.3 nmol cardiolipin).