This review summarizes recent work on the regulation of the permeability transition pore, a cyclosporin A-sensitive mitochondrial channel that may play a role in intracellular calcium homeostasis and in a variety of forms of cell death. The basic bioenergetics aspects of pore modulation are discussed, with some emphasis on the links between oxidative stress and pore dysregulation as a potential cause of mitochondrial dysfunction that may be relevant to cell injury.
A study is presented of the factors affecting the H+/e- stoichiometry of the proton pump of mitochondrial cytochrome c oxidase, isolated and reconstituted in phospholipid vesicles (COV). Under level flow conditions, i.e., in the absence of a transmembrane delta muH+, the H+/e- ratio, obtained from spectrophotometric measurements of the initial rates of electron flow and H+ release specifically elicited by cytochrome c, varied from around 0 to 1, depending on the actual rate of electron flow through the oxidase. At steady state the H+/e- ratio for the oxidase was specifically depressed by the transmembrane delta pH. The study of the H+/e- ratio of the pump was complemented by an analysis of the redox pattern of cytochrome c, CuA, and heme a. From both sets of results and recent structural data from other groups, it is concluded that the dependence of the H+/e- ratio on the rate of electron flow through the oxidase and transmembrane delta pH is associated with the possible occurrence of two electron transfer pathways in cytochrome c oxidase, a coupled one (cyt c-->CuA-->heme a-->heme a3-CuB) and a decoupled one (cyt c-->CuA-->heme a3-CuB). The contributions of the two pathways, differently affected by kinetics and thermodynamic factors, will determine the actual H+/e- ratio of the pump. A possible role of heme a in the proton pump and the physiological implication of the variable H+/e- ratio in the oxidase are discussed.
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. I n 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 proteinldye 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. I n striking contrast with the fragments, no accumulation or spectral shifts of acridines and amino phenazines have been observed with intact mitochondria. On the other hand in the latter case it has been reported that energization brings about accumulation and spectral shifts of ethidium bromide [7-91. Divalent cations also appear to bind to membrane sites of energized mitochondria [lo].We now report that other groups of cationic dyes, such as the safranines and the cyanines, which, differently from the acridines and the aminophenazine and like ethidium bromide, have a permanent charge on the chromophoric group and do not undergo protonation, are taken up through an energylinked process by intact mitochondria. The uptake results in spectral shifts which are interpreted as due 38 Eur. J. Biochem., Vo1.34 to stacking of the dye on membrane sites in low polarity environments.The presence of binding sites on the membrane for divalent cations and cationic dyes are taken to support a mechanism of active transport based on changes of affinity of membrane sites for the translocated species [l I, 121, rather than one based on an electrophoretic process driven by an electrical gradient [I 3 -161.
MATERIALS AND METHODSLiver mitochondria were prepared in 0.25M sucrose, 5 mM Tris-C1 and I mM EDTA, p H 7.4. The final washing was carried out in a n EDTA-free medium. The structural formulae of safranine (I) and pinacyanole (11) used in this study are shown below.The absorbance of the dyes was followed with a dual-wavelength spectrophotometer. The spectra were made either with a noncommercial split-beam spectrophotometer, built in the workshop of the Johnson Foundation (Philadelphia) or with the Hitachi-Perkin Elmer spectrophotometer (Model 124).Oxygen uptake was determined with a Clark electrode sui...
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