Localization of reactive cysteine residues by maleidoyl undecagold in the mitochondrial creatine kinase octamer.
Octamers of mitochondrial creatine kinase (Mi-CK) were modified with the thiol-specific reagents N-ethylmaleimide or the gold-coupled derivative, maleidoyl undecagold. The kinetics of inhibition of the Mi-CK catalysis was shown to be comparable for both reagents, suggesting that the large gold cluster complex is accessible to the reactive cysteines. SDS-PAGE analysis revealed that two of eight cysteines per Mi-CK monomer were labeled with maleidoyl undecagold with a similar affinity for the functional maleimide group. Gel exclusion chromatography of labeled molecules showed that the octameric structure of Mi-CK was preserved after thiol modification. Freeze-dried gold-labeled octamers visualized by electron microscopy under cryo-conditions were enhanced in contrast and showed a well-preserved fourfold symmetry of the end-on view. Image analysis of gold-labeled Mi-CK exhibited an averaged end-on view with four strong contrast signals located at the periphery of the octamer, whereas the center of the molecule remained electron translucent. We conclude that the two cysteine residues per monomer labeled with maleidoyl undecagold are located at the octamer's perimeter and we discuss the possible role of these reactive cysteines in enzyme catalysis.
Schnyder
T
T
Institute of Cell Biology, Swiss Federal Institute of Technology, ETH-Hönggerberg, Zürich.
Tittmann
P
P
Winkler
H
H
Gross
H
H
Wallimann
T
T
D000818:Animals; D002645:Chickens; D002850:Chromatography, Gel; D003402:Creatine Kinase / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure; D003545:Cysteine / Q000032:analysis*; D004591:Electrophoresis, Polyacrylamide Gel; D005033:Ethylmaleimide; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D008854:Microscopy, Electron; D008929:Mitochondria, Heart / Q000201:enzymology*; D050607:Organogold Compounds; D009942:Organometallic Compounds / Q000737:chemistry*
D050607:Organogold Compounds; D009942:Organometallic Compounds; C095517:maleidoyl undecagold; D003402:Creatine Kinase; D003545:Cysteine; D005033:Ethylmaleimide