Oxidative post-translational modification of the brain isoform of creatine kinase: Structural and functional consequences

Oxidative modification of proteins, including modification by the reactive aldehyde 4-hydroxy-2-nonenal (4HNE), has been implicated in the pathogenesis of Alzheimer's disease (AD). Initial reports indicated that the brain isoform of creatine kinase (CK-BB), a cytosolic enzyme that reversibly catalyzes the transfer of the high-energy phosphoryl group from phosphocreatine to MgADP for rapid regeneration of ATP, was oxidized to a greater extent and had decreased activity in AD brain tissues than in age-matched control detected by two dimensional Western blot and activity analysis. As well, behavior studies showed that intake of grape seed extract (GSE) enriched in antioxidant polyphenols enhanced cognition, suggesting the importance of identifying protein changes induced by GSE. Following GSE treatment, two dimensional gel proteomic studies indicated changes in the abundance and isoform complexity of several proteins in rat brain including CK-BB. We carried out experiments, using 4HNE as a model for AD-related oxidative stress, to examine the structural and functional consequences of in vitro 4HNE modification of recombinant human CK-BB. 4HNE modifications were localized by Fourier transform-ion cyclotron resonance mass spectrometry (FT-ICR MS) and active site modifications were correlated with decreased activity. We determined that the abundance of CK-BB per mg 16,000 x g supernate protein was decreased by approximately 80% in AD compared to age-matched control frontal cortex, however, CK-BB specific activity was equivalent. We optimized a purification protocol for CK-BB from human brain samples with approximately 67% recovery and analyzed for oxidative modifications by FT-ICR MS. The purified CK-BB did not contain any detectable 4HNE adducts; however, both the AD and age-matched control CK-BB had methionine and tryptophan oxidations which apparently did not affect CK-BB activity. The specific activity of CK-BB associated with the 16,000 x g particulate fraction, however, was reduced in AD to 36% of age-matched control. This report is the first systematic analysis of the structural and the functional consequences of in vitro 4HNE modification of CK-BB. In addition, it represents the first thorough analysis of specific activity, abundance, and oxidative modifications of CK-BB from AD and age-matched control frontal cortex 16,000 x g supernate and particulate fractions.