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KMID : 0617320000090010081
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Journal of Pharmacetical Sceiences Ewha Womans University
2000 Volume.9 No. 1 p.81 ~ p.88
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Oxidative Modification of Nucleoside Diphosphate Kinase and Its Identification by Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry
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Song, Eun Joo
Kim, Yang Sun/Chung, Ji Young/Kim, Eunhee/Chae, Suhn-Kee/Lee, Kong-Joo
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Abstract
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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_2O_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_2O_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_2O_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.
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