Abstract
Recent observations point to the role played by Zn2+ as an inducer of neuronal death. Two Zn2+ targets have been identified that result in inhibition of mitochondrial respiration: the bc1 center and, more recently, alpha-ketoglutarate dehydrogenase. Zn2+ is also a mediator of oxidative stress, leading to mitochondrial failure, release of apoptotic peptides, and neuronal death. We now present evidence, by means of direct biochemical assays, that Zn2+ is imported through the Ca2+ uniporter and directly targets major enzymes of energy production (lipoamide dehydrogenase) and antioxidant defense (thioredoxin reductase and glutathione reductase). We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2+ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2+ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2+ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 microm Zn2+. An additional, unidentified import route functions at higher Zn2+ concentrations. (g) No enzyme inactivation is observed for Ca2+-induced MPT. These observations strongly suggest that, unlike Ca2+, exogenous Zn2+ interferes with mitochondrial NADH production and directly alters redox protection in the matrix, contributing to mitochondrial dysfunction. Inactivation of these enzymes by Zn2+ is irreversible, and thus only their de novo synthesis can restore function, which may underlie persistent loss of oxidative carbohydrate metabolism following transient ischemia.
Highlights
A growing number of reports have linked changes in intracellular free Zn2ϩ to pathological processes, in the nervous system [1]
We demonstrate the following. (a) These matrix enzymes are rapidly inhibited by application of Zn2؉ to intact mitochondria. (b) Delayed treatment with membrane-impermeable chelators has no effect, indicating rapid transport of biologically relevant quantities of Zn2؉ into the matrix. (c) Membrane-permeable chelators stop but do not reverse enzyme inactivation. (d) Enzyme inhibition is rapid and irreversible and precedes the major changes associated with the mitochondrial permeability transition (MPT). (e) The extent and rate of enzyme inactivation linearly correlates with the MPT onset and propagation. (f) The Ca2؉ uniporter blocker, Ruthenium Red, protects enzyme activities and delays pore opening up to 2 M Zn2؉
Description of Microplate-based Ca2ϩ and Zn2ϩ Swelling Titrations—The MPT is commonly monitored as changes in light scattering, which is especially pronounced for liver mitochondria
Summary
A growing number of reports have linked changes in intracellular free Zn2ϩ to pathological processes, in the nervous system [1]. We employed analysis of the combined effects of Ca2ϩ and Zn2ϩ on the time course of mitochondrial swelling, whereas varying the concentrations of both metals. If the effectors (i.e. Ca2ϩ and Zn2ϩ) compete for a single “physical site” (which in the case of MPT may be the Ca2ϩ uniporter, a component of the permeability pore, or an unknown matrix enzyme/protein), this site can be completely occupied by saturating concentrations of either metal.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.