Oxidative modification of nucleoside diphosphate kinase and its identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.
Nucleoside diphosphate kinase (NDPK, Nm23) has been implicated as a multifunctional protein. However, the regulatory mechanism of NDPK is poorly understood. We have examined the modification of NDPK in oxidative stresses. We found that oxidative stresses including diamide and H(2)O(2) treatment cause disulfide cross-linking of NDPK inside cells. This cross-linking was reversible in response to mild oxidative stress, and irreversible to strong stress. This suggests that disulfide cross-linked NDPK may be a possible mechanism in the modification of cellular regulation. To confirm this idea, oxidative modification of NDPK has been performed in vitro using purified human NDPK H(2)O(2) inactivated the nucleoside diphosphate (NDP) kinase activity of NDPK by producing intermolecular disulfide bonds. Disulfide cross-linking of NDPK also dissociated the native hexameric structure into a dimeric form. The oxidation sites were identified by the analysis of tryptic peptides of oxidized NDPK, using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Intermolecular cross-linking between Cys109-Cys109, which is highly possible based on the X-ray crystal structure of NDPK-A, and oxidations of four methionine residues were identified in H(2)O(2)-treated NDPK. This cross-linkng was confirmed using mutant C109A (NDPK-A(C109A)) which had similar enzymatic activity as a wild NDPK-A. Mutant NDPK-A(C109A) was not cross-linked and was not easily denatured by the oxidant. Therefore, enzymatic activity and the quaternary structure of NDPK appear to be regulated by cross-linking with oxidant. These findings suggest one of the regulatory mechanisms of NDPK in various cellular processes.
Song
E J
EJ
Center for Cell Signaling Research, Division of Molecular Life Sciences and College of Pharmacy, Ewha Womans University, Seoul, Korea.
Kim
Y S
YS
Chung
J Y
JY
Kim
E
E
Chae
S K
SK
Lee
K J
KJ
D000595:Amino Acid Sequence; D000818:Animals; D003432:Cross-Linking Reagents; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014407:Tumor Cells, Cultured
D003432:Cross-Linking Reagents; D010446:Peptide Fragments; D009701:Nucleoside-Diphosphate Kinase; D003545:Cysteine