Abstract
Protein oxidation has detrimental effects on the brain functioning, which involves inhibition of the crucial enzyme, brain type creatine kinase (CKBB), responsible for the CK/phosphocreatine shuttle system. Here we demonstrate a susceptibility of CKBB to several ordinary stressors. In our study enzymatic activity of purified recombinant brain-type creatine kinase was evaluated. We assayed 30 nMconcentration of CKBB under normal and stress conditions. In the direction of phosphocreatine formation hydrogen peroxide and heat treatments altered CKBB activity down to 26 and 14%, respectively. Also, examination of immunoblotted membrane patterns by SDS-PAGE electrophoresis and western blot analysis showed a decrease in expression levels of intrinsic CKBB enzyme in HeLa andA549 cells. Hence, our results clearly show that cytosolic CKBB is extremely sensitive to oxidative stress and heat induced inactivation. Therefore, due to its susceptibility, this enzyme may be defined as a potential target in brain damage.
Highlights
A mple energy supply is crucial for proper functioning of the brain tissue
Since CKBB is differentially expressed and critical for ATP metabolism, we examined enzyme’s potential to retain kinase activity affected by various stresses
Studies on human muscle creatine kinase have confirmed that thermal irreversibility of the enzyme was reached under the temperature of 56 °C [2]
Summary
Overexpression of CKBB in mammalian cell lines. For the in vitro detection of CKBB in HeLa and A549 cell we transfected cells with plasmids pCMV-Flag carrying CKBB gene insert. The plasmids harboring Flag-CKBB was transiently transfected into 60 mm dish cultured with A549 or HeLa cells using Lipofectamine/Plus Reagents (Invitrogen) according to the manufacture’s protocol. The plasmids harboring Flag-CKBB, constructs were transiently transfected into 60 mm dish cultured with A549 or HeLa cells using Lipofectamine/Plus Reagents (Invitrogen) according to the manufacture’s protocol. The gel was either stained in a Coomassie Brilliant Blue solution or immunoblotted with specific antibodies. Recombinant CKBB was overexpressed and purified according to the procedures described in the previous study [17]. CKBB activity was determined in the forward direction (phosphocreatine formation) according to the pH-colorimetry method [18] with some modifications
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