Mitochondrial Creatine Kinase Is a Prime Target of Peroxynitrite-induced Modification and Inactivation

Abstract

The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creatine kinase (Mib-CK; EC 2.7.3.2) was observed at different stages of complexity (i) with purified Mi-CK, (ii) with enzyme bound on isolated mitoplasts, and (iii) within intact respiring mitochondria. Creatine-stimulated respiration was abolished by PN concentrations likely to be physiological and far before the respiratory chain itself was affected, thus demonstrating that Mi-CK is a prime target for inactivation by PN in intact mitochondria. The inactivation by PN of Mi-CK was reversed by 22% with 2-mercaptoethanol. More remarkable protective effects were noticed with the full set of CK substrates, e.g. 30 and 50% protection with MgATP plus creatine and MgADP plus phosphocreatine, respectively, but not with each substrate alone. These data indicate an involvement of the active-site Cys-278 residue of Mi-CK in this process. Furthermore, changes in endogenous tryptophan fluorescence intensity and spectral changes after reaction of Mi-CK with PN suggest additional modifications of Trp and Tyr residues. PN-inactivated Mi-CK can no longer be efficiently converted into dimers by incubation with reagents inducing a transition state analog complex at the active site. Thus, obviously, upon reaction of octameric Mi-CK with PN, the octamer-dimer equilibrium of Mi-CK is also affected. The consequences for cellular energy homeostasis and calcium handling are discussed.

Peroxynitrite Irreversibly Inactivates the Human Xenobioticmetabolizing Enzyme Arylamine N-Acetyltransferase 1 (NAT1) in Human Breast Cancer Cells

“…49), but concentrations of up to 500 M have also been reported Ref. 50). The rate constant for the inactivation of recombinant NAT1 by peroxynitrite was determined to be 5 ϫ 10 4 M Ϫ1 ⅐s Ϫ1 , indicating that the reaction between peroxynitrite and the enzyme is extremely fast.…”

“…SIN1 has been shown to attack many biological targets in the same manner as authentic peroxynitrite and, thus, has been widely used as a source of peroxynitrite in studies using purified proteins and cultured cells (47). Physiological concentrations of peroxynitrite in vivo have been estimated to be ϳ50 M (49), although 500 M concentrations have been found within phagolysosomes of activated macrophages (50).…”

Cigarette Smoke Exposure and Hypercholesterolemia Increase Mitochondrial Damage in Cardiovascular Tissues

“…Several studies have shown that oxidative stress is associated with increased mitochondrial DNA damage, lipid peroxidation, inhibition of electron transport, and inactivation of specific mitochondrial enzymes. 13,[27][28][29][30] The mitochondrial damage associated with SHS exposure and hypercholesterolemia reported here may compromise important metabolic processes that influence both endothelial and vascular smooth muscle cell function, key components of atherogenesis. Consequently, our findings are consistent with present theories that oxidative stress mediates CVD by causing mitochondrial damage and dysfunction in tissues, which ultimately lead to compromised energetic capacities and cell dysfunction, important early events in CVD.…”

Proteomic identification of brain proteins in the canine model of human aging following a long-term treatment with antioxidants and a program of behavioral enrichment: Relevance to Alzheimer's disease

“…CKs are prime targets of oxidative damage. MtCK in particular is a principal target of such damage, not only because if its sensitivity [66,100], but also due to its mitochondrial localization. Our laboratory has shown that though there is increased expression of CK in AD, it is significantly oxidized and its activity significantly reduced [7,33].…”