The Problem of Cation‐Binding Sites in the Energized Membrane of Intact Mitochondria

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Abstract
Weak bases, such as the acridine and the aminophenazine dyes, which undergo protonation in the chromophoric group, are actively bound in sonicated fragments. On the other hand strong bases such as the divalent cations, the safranine and the cyanine dyes, which carry a permanent charge, are actively bound in intact mitochondria. In the latter case the process results in stimulation of the respiration, H+ ejection, and a large change in absorbance, which is attributed to a stacking of the dyes. The effects are inhibited or reversed by uncouplers, and insensitive to nigericin.Four alternatives are considered: accumulation in the inner aqueous space; binding to matrix proteins; binding to the outer membrane surface; and binding to sites in the membrane fabric. The view that the energized membrane contains high affinity sites for cationic dyes is supported by: comparison of the spectra of the membrane‐bound and polyanion‐bound dye; competition with divalent cations; osmotic activity; and dependence of the spectral shift on the protein/dye ratio. The data are in accord with the concept that the mitochondrial membrane acts as a fixed negative‐charge system where electrostatic interactions occur in microenvironments of low polarity.