Inactivation of creatine kinase by S-glutathionylation of the active-site cysteine residue

Sharanya Reddy, A. Daniel Jones, Carroll E Cross, Patrick S Y Wong, Albert Van Der Vliet

Research output: Contribution to journalArticle

103 Scopus citations

Abstract

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 (Cys283) 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.

Original languageEnglish (US)
Pages (from-to)821-827
Number of pages7
JournalBiochemical Journal
Volume347
Issue number3
DOIs
StatePublished - May 1 2000

Keywords

  • Glutathione
  • MALDI mass spectrometry
  • Oxidative stress
  • S-thiolation

ASJC Scopus subject areas

  • Biochemistry

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