A thiol oxidation interpretation of the Cu 2+ effects on rat liver mitochondria

Abstract

A comparative study of the Cu 2+ effects, binding and reduction, has been performed on rat liver mitochondria. In the first minutes, Cu 2+ (≤ 50 μm) is massively bound and reduced to the extent of 70%–80% while a simultaneous activation of respiration takes place. Then the remaining 20% or so of Cu 2+ are progressively bound and reduced while respiratory inhibition, Ca 2+ and Mg 2+ effluxes, and swelling are observed. EDTA, used as a copper chelator, prevents or reduces the copper effects and removes part of the bound copper, according to the time of introduction in the incubation medium after Cu 2+. The results sugest that the two steps of the copper binding and the effects following involve mainly first the outer (cytosol side) proteins of the inner membrane and then those of the inner membrane. 100 μM dithiothreitol and 100 μM glutathione used as antioxidant thiol reagents prevent, as does EDTA, but do not reverse the 25 μM copper effects. They also decrease the copper binding; however, no relationship between binding and preventive action is observed. It is shown that glutathione and dithiothreitol have a specific potent ability to reduce Cu 2+ which explains that in presence of these reagents copper may react with mitochondria partly or entirely in the form of Cu +. These findings suggest that Cu 2+ in its Cu + form has no mitochondrial effect. A mechanism of copper action involving oxidation of some membrane thiol groups is discussed.