Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue.
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 ionization MS confirmed that the molecular mass of CK was increased, consistent with addition of one GSH molecule/molecule of CK. Using trypsin digestion, HPLC and MS analysis, the active-site cysteine residue (Cys(283)) was identified as the site of thiolation. Reversal of thiolation was shown to be rapid when GSH is abundant, rendering dethiolation of CK thermodynamically favoured within the cell. We conclude that S-glutathionylation of CK could be one mechanism to explain temporary reversible loss in activity of CK during ischaemic injury. The maintainance of GSH levels represents an important mechanism for regeneration of active CK from S-glutathionylated CK.
Reddy
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S
Department of Internal Medicine and Center for Comparative Lung Biology and Medicine, University of California, Davis, CA 95616, USA. sreddy@ucdavis.edu
Jones
A D
AD
Cross
C E
CE
Wong
P S
PS
Van Der Vliet
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003402:Creatine Kinase / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D004229:Dithiothreitol / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D019803:Glutathione Disulfide / Q000378:metabolism; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000201:enzymology; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000378:metabolism; D011817:Rabbits; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013816:Thermodynamics; D014357:Trypsin / Q000378:metabolism
D007527:Isoenzymes; D010446:Peptide Fragments; D013438:Sulfhydryl Compounds; D003402:Creatine Kinase; D014357:Trypsin; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; D019803:Glutathione Disulfide