Reactivation of creatine kinase by dithiothreitol prior to use in an in vitro translation extract.
BACKGROUND
In vitro protein synthesis on exogenous messenger ribonucleic acids can be performed in various systems including cytoplasmic extract from eukaryotic cells, rabbit reticulocyte lysate and wheat germ extract. For optimal translation, an energy regeneration system based on creatine phosphate and creatine kinase is commonly employed for the regeneration of the endogenous adenosine triphosphate pools. Creatine kinase purchased from various commercial suppliers can be partially oxidised. Oxidised creatine kinase is not biologically active and might not allow the efficient initiation of translation of exogenous mRNAs in eukaryotic cell extracts in vitro.


RESULTS
We successfully used dithiothreitol to reduce and therefore reactivate commercially available creatine kinase. When employed in cytoplasmic extracts obtained from eukaryotic cells grown in monolayers, the reactivated creatine kinase restored translation of the exogenous mRNAs.


CONCLUSIONS
Lyophilised creatine kinase obtained from commercial suppliers can be purchased as an oxidised monomer. The reactivation of creatine kinase using a reducing agent such as dithiothreitol restores the biological activity of this enzyme. This procedure might therefore be extended to various other in vitro conditions and biological systems in which the maintenance of an efficient ATP-regenerating system is critical.
Favre
Daniel
D
Division Gastroenterology and Hepatology, Department of Internal Medicine, University Hospital of Zurich, Switzerland. Daniel.Favre@usz.ch
Muellhaupt
Beat
B
D000818:Animals; D002460:Cell Line; D002474:Cell-Free System / Q000201:enzymology; D003402:Creatine Kinase / Q000187:drug effects / Q000378:metabolism*; D006224:Cricetinae; D004229:Dithiothreitol / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects; D004793:Enzyme Reactivators / Q000494:pharmacology*; D006801:Humans; D010084:Oxidation-Reduction; D010936:Plant Extracts; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D014908:Triticum
D004793:Enzyme Reactivators; D010936:Plant Extracts; D012333:RNA, Messenger; D003402:Creatine Kinase; D004229:Dithiothreitol