Disulfide bond within mu-calpain active site inhibits activity and autolysis.
Oxidative processes have the ability to influence mu-calpain activity. In the present study the influence of oxidation on activity and autolysis of mu-calpain was examined. Furthermore, LC-MS/MS analysis was employed to identify and characterize protein modifications caused by oxidation. The results revealed that the activity of mu-calpain is diminished by oxidation with H2O2 in a reversible manner involving cysteine and that the rate of autolysis of mu-calpain concomitantly slowed. The LC-MS/MS analysis of the oxidized mu-calpain revealed that the amino acid residues 105-133 contained a disulfide bond between Cys(108) and Cys(115). The finding that the active site cysteine in mu-calpain is able to form a disulfide bond has, to our knowledge, not been reported before. This could be part of a unique oxidation mechanism for mu-calpain. The results also showed that the formation of the disulfide bond is limited in the control (no oxidant added), and further limited in a concentration-dependent manner when beta-mercaptoethanol is added. However, the disulfide bond is still present to some extent in all conditions indicating that the active site cysteine is potentially highly susceptible to the formation of this intramolecular disulfide bond.
Lametsch
René
R
Department of Food Science, University of Copenhagen, 1958 Frederiksberg C, Denmark.
Lonergan
Steven
S
Huff-Lonergan
Elisabeth
E
D000818:Animals; D001329:Autolysis; D002154:Calpain / Q000737:chemistry* / Q000378:metabolism*; D020134:Catalytic Domain; D002853:Chromatography, Liquid; D004220:Disulfides / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D066298:In Vitro Techniques; D008623:Mercaptoethanol / Q000494:pharmacology; D008958:Models, Molecular; D018482:Muscle, Skeletal / Q000378:metabolism; D010084:Oxidation-Reduction; D013552:Swine; D053719:Tandem Mass Spectrometry
D004220:Disulfides; D008623:Mercaptoethanol; D006861:Hydrogen Peroxide; D002154:Calpain; C449002:mu-calpain