Free radical induced inactivation of creatine kinase: sites of interaction, protection, and recovery (original) (raw)
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Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue
Biochemical Journal, 2000
Protein S-thiolation, the formation of mixed disulphides of cysteine residues in proteins with low-molecular-mass thiols, occurs under conditions associated with oxidative stress and can lead to modification of protein function. In the present study, we examined the site of S-thiolation of the enzyme creatine kinase (CK), an important source of ATP in myocytes. Inactivation of this enzyme is thought to play a critical role in cardiac injury during oxidative stress, such as during reperfusion injury. Reaction of rabbit CK M isoenzyme with GSSG, used to model protein S-thiolation, was found to result in enzyme inactivation that could be reversed by GSH or dithiothreitol. Measurement of GSH that is released during the thiolation reaction indicated that the maximum extent of CK thiolation was approx. 1 mol of GSH\mol of protein, suggesting thiolation on one reactive cysteine residue. Accordingly, matrix-assisted laser-desorption
Journal of Molecular and Cellular Cardiology, 1985
The objective of this study was to test the hypothesis that cytotoxic oxygen metabolites participate in lytic cardiac cell damage, detected as creatine kinase release, upon reoxygenation of hypoxic, isolated buffer-perfused hearts (oxygen paradox). Perfusate additives included: superoxide dismutase (30 mg/1) ; catalase (2 rag/l) ; deferoxamine (0.5 mM) ; and allopurinol (1 mM). Creatine kinase release upon reoxygenation was reduced, to levels not significantly different from nonhypoxic controls, by adding either catalase, allopurinol or deferoxamine to the buffer during hypoxia. Reduced creatine kinase leakage was not accompanied by parallel preservation of ventricular function or coronary vascular resistance. Administration of catalase during hypoxia was superior to administering it only during reoxygenation. Treatment with catalase during both hypoxia and reoxygenation provided no more protection than administration only during hypoxia. The data suggest that an important component of hypoxia-indueed cardiac cell damage is due primarily to hydrogen peroxide, which may then form hydroxyl radical. Superoxide anion plays an important role as a precursor of these species, but added superoxide dismutase alone did not significantly reduce creatine kinase loss. The data also suggest that damage resulting in creatine kinase release upon reoxygenation occurs during oxygen deprivation, and it is mediated in part by cytotoxic oxygen metabolites.
Mitochondrial Creatine Kinase Activity Prevents Reactive Oxygen Species Generation
Journal of Biological Chemistry, 2006
mitochondrial hexokinase activity (mt-HK) plays a preventive antioxidant role because of steady-state ADP recycling through the inner mitochondrial membrane in rat brain. In the present work we show that ADP recycling accomplished by the mitochondrial creatine kinase (mt-CK) regulates reactive oxygen species (ROS) generation, particularly in high glucose concentrations. Activation of mt-CK by creatine (Cr) and ATP or ADP, induced a state 3-like respiration in isolated brain mitochondria and prevention of H 2 O 2 production obeyed the steady-state kinetics of the enzyme to phosphorylate Cr. The extension of the preventive antioxidant role of mt-CK depended on the phosphocreatine (PCr)/Cr ratio. Rat liver mitochondria, which lack mt-CK activity, only reduced state 4-induced H 2 O 2 generation when 1 order of magnitude more exogenous CK activity was added to the medium. Simulation of hyperglycemic conditions, by the inclusion of glucose 6-phosphate in mitochondria performing 2-deoxyglucose phosphorylation via mt-HK, induced H 2 O 2 production in a Crsensitive manner. Simulation of hyperglycemia in embryonic rat brain cortical neurons increased both ⌬⌿ m and ROS production and both parameters were decreased by the previous inclusion of Cr. Taken together, the results presented here indicate that mitochondrial kinase activity performed a key role as a preventive antioxidant against oxidative stress, reducing mitochondrial ROS generation through an ADP-recycling mechanism.
Journal of Biological Chemistry, 1998
The reaction of peroxynitrite (PN) with sarcomeric mitochondrial creatine kinase (Mi b -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.
Hydroxyl radical oxidation of cytochrome c by aerobic radiolysis
Free Radical Biology and Medicine, 2004
The reaction of radiolytically generated S OH with cytochrome c was investigated by mass spectrometry. Tryptic digestion and characterization of the oxidized peptides by MALDI-TOF and ESI tandem mass spectrometry identified eight different amino acid residues with oxidized side chains with no cleavage of the protein detected. Solvent-accessible aromatic and methionine residues are the most susceptible to oxidation by S OH. These results support the careful use of S OH in characterizing protein surfaces. Dose-response studies identified the residues most prone to oxidation to be Phe-36, Phe-46, and Met-80. Hydroxylation of Phe-36 and Phe-46 should serve as indicators of the presence of S OH in the mitochondrial intermembrane space. Using solutions containing 50 at.% 18 O, our study also provides a novel method of determining the source of oxygen during S OH-mediated oxidation of proteins and contributes to identification of the modified residue type, with PheNTyrNMet in 18 O incorporation. During aerobic radiolysis, UV-vis spectroscopy indicates that ferrocytochrome c reaches a steady state concomitant with reduction of the heme.
The Journal of biological chemistry, 1987
In most cases the apparent target size obtained by radiation inactivation analysis corresponds to the subunit size or to the size of a multimeric complex. In this report, we examined whether the larger than expected target sizes of some enzymes could be due to secondary effects of free radicals. To test this proposal we carried out radiation inactivation analysis on Escherichia coli DNA polymerase I, Torula yeast glucose-6-phosphate dehydrogenase, Chlorella vulgaris nitrate reductase, and chicken liver sulfite oxidase in the presence and absence of free radical scavengers (benzoic acid and mannitol). In the presence of free radical scavengers, inactivation curves are shifted toward higher radiation doses. Plots of scavenger concentration versus enzyme activity showed that the protective effect of benzoic acid reached a maximum at 25 mM then declined. Mannitol alone had little effect, but appeared to broaden the maximum protective range of benzoic acid relative to concentration. The ...
Journal of Molecular and Cellular Cardiology, 1998
The importance of mitochondrial creatine kinase (mi-CK) in oxidative muscle was tested by studying the functional properties of in situ mitochondria in saponinskinned muscle fibres from sarcomeric mi-CK-deficient (mutant) mice. Biochemical analyses showed that the lack of mi-CK in mutant muscle was associated with a decrease in specific activity of MM-CK in mutant ventricle, and increase in mutant soleus (oxidative) muscle. Lactate dehydrogenase activity and isoenzyme analysis showed an increased glycolytic metabolism in mutant soleus. No change was observed in ventricular muscle. In control animals, the apparent K m of mitochondrial respiration for ADP in ventricle and soleus (232±36 and 381±63 , respectively) was significantly reduced in the presence of creatine (52±8 and 45±12 , respectively). There was no change in the K m in oxidative fibres from mutant mice (258±27 and 399±66 , respectively) compared with control, though surprisingly, it was also significantly decreased in the presence of creatine (144±8 and 150±27 , respectively) despite the absence of mi-CK. It is proposed that in mutant (and perhaps normal) oxidative tissue, cytosolic MM-CK can relocate to the outer mitochondrial membrane, where it is coupled to oxidative phosphorylation by close proximity to porin, and the adenine nucleotide translocase. Such an effect can preserve the functioning of the CK shuttle and the energetic properties of mi-CK deficient tissue.
Creatine kinase: The reactive cysteine is required for synergism but is nonessential for catalysis
Biochemistry, 1993
Chemical modification of rabbit muscle creatine kinase (CK) with thiol-specific reagents led to partial or complete inactivation of the enzyme. Using site-directed mutagenesis, we have substituted the corresponding reactive Cys278 in the chicken cardiac mitochondrial creatine kinase (Mib-CK) with either glycine, serine, alanine, asparagine, or aspartate. The resulting mutant Mib-CK enzymes showed qualitatively similar changes in their enzymatic properties. In both directions of the CK reaction, a shift of the pH optimum to lower values was observed. Mutant Mib-CKs were severalfold more sensitive to inhibition by free ADP in the reverse reaction (ATP synthesis) and to free ATP in the forward reaction (phosphocreatine synthesis). With the exception of C278D, all mutant enzymes were specifically activated by chloride and bromide anions. C278D and wild-type Mib-CK were significantly inhibited under the same conditions. At low chloride concentrations, the V,,, of C278D was about 12-fold higher than that of C278N. Thus, Cys278 probably provides a negative charge which is directly or indirectly involved in maximizing CK activity. Under near-optimal conditions in the reverse reaction, mutants C278G and C278S showed about an 1 l-fold increase inKm(PCr), but only 1.7-and 2.8-fold reductions in V-, respectively, compared to wild-type Mib-CK. Thus, the reactive cysteine clearly is not essential for catalysis. For rabbit muscle CK, substrate binding had been shown to be synergistic (Le., Kd > K,,,). We confirmed this finding for wild-type Mib-CK by determining the Kd and K m values for both substrates in the forward reaction. Analysis of these constants for the two mutant enzymes C278G and C278S showed that the reactive cysteine (1) is not directly involved in binding either substrate (&values for mutants were not dramatically changed compared to the wild type) and is necessary for synergistic substrate binding (Kd values for mutants were smaller than the corresponding Km values). These results suggest that the reactive cysteine is necessary to confer conformational changes upon substrate binding and support the proposal that this residue has a role in shaping the active site, possibly by acting as a hinge between the two substrate binding sites. * Corresponding author. Telephone (01) 377 3447, FAX (01) 371 2894. 1 Abbreviations: Ac, acetate anion; CK, creatine kinase; B-CK, braintype C K M-CK, muscle-type CK; Mi-CK, mitochondrial-type CK isoforms; Mib-CK, basic, "sarcomeric" Mi-CK isoform; PCr, phosphocreatine; Cr, creatine. Mutants with amino acid substitutions are denoted in the standard one-letter code by the wild-type residue and numbered position within the sequence, followed by the amino acid substitution. Thus, C278G Mib-CK is a mutant enzyme with a glycine residue at position 278 instead of thecysteine. Kd refers to the dissociation constant of the reaction E + A -EA or E + B -EB, and K m refers to the Michaelis-Menten constant of the reaction EA + B -EAB or EB + A -EAB, whereby A and B represent either ADP and PCr or ATP and Cr, respectively. Forward reaction is PCr synthesis; reverse reaction is ATP synthesis.