Ibolya Schmehl

Double-stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin

The transport of genetic material across biomembranes is a process of great relevance for several fields of study. However, much remains to be learned about the mechanisms underlying transport, one of which implies the involvement of proteic DNA‐conducting pores. Entry of genetic material into mitochondria has been observed under both physiological and pathological conditions. We report here that double‐stranded DNA can move through a planar bilayer membrane containing isolated mitochondrial porin (voltage‐dependent anion channel). The transport is driven by the applied electrical field, and the presence of DNA is associated with a decrease of current conduction by the pores. The passage of DNA does not take place if the bilayer has not been doped with any protein or in the presence of both reconstituted porin and anti‐porin antibody. Translocation does not occur if the bilayer contains Shigella sonnei maltoporin, gramicidin A channels, or a 30 pS anion‐selective channel plus other proteins. These results show that mitochondrial porin is capable of mediating the transport of genetic material, revealing a new property of this molecule and futher confirming the idea that DNA can move through proteic pores.—Szabò, I., Bàthori, G., Tombola, F., Coppola, A., Schmehl, I., Brini, M., Ghazi, A., De Pinto, V., Zoratti, M. Double‐stranded DNA can be translocated across a planar membrane containing purified mitochondrial porin. FASEB J. 12, 495–502 (1998)

Nature of respiratory stimulation in hyperthyroidism: the redox behaviour of cytochrome c

Hyperthyroid mitochondria show an increased K m and V max in the high affinity phase of cytochrome oxidase kinetics. During inhibitor titrations, cytochrome c shows a different redox behaviour in hyperthyroid with respect to protonophore‐treated euthyroid mitochondria. The observations are discussed in terms of a different regulation of electron input and output into the respiratory chain during slip and leak types of uncoupling. In hyperthyroid mitochondria during inhibitor titrations, the pattern of the relationship between uncoupler‐induced extra‐respiration and membrane potential is highly non‐linear. The complex nature of the respiratory stimulation in hyperthyroid mitochondria is discussed.

Activation of respiration and loss of thermodynamic control in hyperthyroidism Is it due to increased slipping in mitochondrial proton pumps

T3 administration increases the extent of non‐linearity in the flow—force relationship between pump proton conductance and protonmotive force. The effect is present also at the ATPase proton pump. These effects are not accompanied by changes in passive proton conductance. Incubation of mitocondria at 45°C also causes an increased non‐linearity, accompanied by a partial increase of proton conductance. It appears that the increase of respiratory activity following T3 administration is due to loss of thermodynamic control within or at the proton pumps, an effect which might be attributed to increased slipping.

The effect of the protonmotive force on the redox state of mitochondrial cytochromes

In the absence of kinetic limitations, as determined either by high substrate concentrations or by absence of respiratory chain inhibitors, we have observed that: (a) the relationship between the percentage reduction of the cytochromes and the protonmotive force is linear in the case of cytochrome c and biphasic in the case of cytochrome b, (b) the redox state of cytochrome c depends only on the membrane potential and not on the total proton motive force and (c) the alkalinization of the matrix enhances the extent of cytochrome c reduction because of the marked inhibitory effect on the cytochrome oxidase activity. Thus, although the redox states of the b, c and aa3 mitochondrial cytochromes depend on the protonmotive force, the quantitative correlation between the two parameters and the relative effects of the electrical and chemical components of the force differ among the various cytochromes.

The effect of respiration on the permeability of the mitochondrial membrane to ions

1. The rates of cation uptake, for either organic cations such as tetrapropylammonium, TPA+, at variable tetraphenylboron concentrations, TPB-, or inorganic cations such as Mn2+, or K+ plus valinomycin, have been measured in mitochondria either respiring, under uncoupler titrations, or non-respiring, under variable K+ diffusion potentials. 2. The flow-force relationship for the respiration-coupled ion fluxes during titrations with uncouplers is almost identical to that obtained for the K(+)-diffusion driven fluxes. Similar results are obtained when TPA+ is replaced with inorganic cations, either monovalent such as K+ (+valinomycin), or divalent such as Mn2+. 3. By applying the Eyring analysis, as developed by Garlid et al. (Garlid, K.D., Beavis, A.D. and Ratkje, S.K. (1989) Biochim. Biophys. Acta 976, 109-121), from the flux-voltage relationships the values for the permeability coefficients and for the energy barriers have been obtained for the transport of the ion pair TPA(+)-TPB-, of Mn2+ and of K+ plus valinomycin, in non-respiring and in respiring, coupled and uncoupled, mitochondria. 4. The findings that the rates of respiration-coupled ion fluxes, at all values of membrane potential, are similar to the rates of the K+ diffusion potential-coupled ion fluxes and the similar pattern of the flux-voltage relationships during the titrations with uncouplers and artificial gradients indicate that the membrane permeability for ions is not modified by respiration.

The Respiratory Stimulation of Hyperthyroid Mitochondria

The respiratory stimulation of hyperthyroid mitochondria, as well as that due to temperature or chloroform in euthyroid mitochondria, has been analyzed by means of the relationsip between the uncoupler-induced extra respiration and membrane potential. We found that: i) the respiratory stimulation in hyperthyroid or chloroform-supplemented mitochondria, as well as the resting respiration at low temperature, are mainly due to pump slip, while ii) the respiratory stimulation at high temperature is largely due to membrane proton leak.

The Redox State of Cytochromes as a New Tool to Assess the Thermodynamic State of Mitochondria

In the present work we have followed the changes of the absorption spectra of mitochondrial suspensions during transitions from anaerobiosis to steady state and after addition of uncouplers. From these spectra we have selected proper wavelengths to follow the kinetics of the spectroscopic changes of the various cytochrome components of the redox chain. We focused our attention to a group of cytochromes, those of the cc1, providing a response similar in significance to that of the electrodes or of the dyes assessing the membrane potential.