title: Oxidative modification of a carboxyl-terminal vicinal methionine in calmodulin by hydrogen peroxide inhibits calmodulin-dependent activation of the plasma membrane Ca-ATPase.
abstract: In order to investigate the possibility that calmodulin (CaM) may be a principal target of reactive oxygen species (ROS) produced under conditions of oxidative stress, we have examined wheat germ CaM for the presence of highly reactive sites that correlate with the loss of function. Using reversed-phase HPLC and FAB mass spectrometry after proteolytic digestion, we have identified the sites of modification by hydrogen peroxide. We find that one of the vicinal methionines (i.e., Met146 or Met147) near the C-terminus of CaM is selectively oxidized. The ability of CaM to bind and to activate the plasma membrane (PM)-Ca-ATPase from erythrocytes was measured. There is a 30-fold decrease in the calcium affinity of oxidatively modified CaM. While there is a little change in the binding constant between the carboxyl-terminal domain of calcium-saturated CaM and a peptide homologous to the autoinhibitory sequence of the PM-Ca-ATPase, we find that there is a 9-fold reduction in the affinity of the amino-terminal domain of CaM with respect to the ability to bind target peptides. The extent of oxidative modification to one of the vicinal methionines near the carboxyl-terminal domain correlates with the loss of CaM-dependent activation of the PM-Ca-ATPase. The presence of oxidatively modified CaM prevents native CaM from activating the PM-Ca-ATPase, indicating that the oxidatively modified CaM binds to the autoinhibitory sequence on the Ca-ATPase in an altered nonproductive conformation. We suggest that the functional sensitivity of CaM to the oxidation of one of the C-terminal vicinal methionines permits CAM to serve a regulatory role in modulating cellular metabolism under conditions of oxidative stress. The predominant oxidation of a methionine near the carboxyl terminal of CaM is rationalized in terms of the enhanced solvent accessibility of these vicinal methionines.
authors.affiliation: Department of Biochemistry, University of Kansas, Lawrence, Kansas 66045-2106, USA.
authors.forename: Y
authors.initials: Y
authors.lastname: Yao
authors.forename: D
authors.initials: D
authors.lastname: Yin
authors.forename: G S
authors.initials: GS
authors.lastname: Jas
authors.forename: K
authors.initials: K
authors.lastname: Kuczer
authors.forename: T D
authors.initials: TD
authors.lastname: Williams
authors.forename: C
authors.initials: C
authors.lastname: Schöneich
authors.forename: T C
authors.initials: TC
authors.lastname: Squier
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000201:enzymology; D002851:Chromatography, High Pressure Liquid; D004789:Enzyme Activation; D006861:Hydrogen Peroxide; D006863:Hydrogen-Ion Concentration; D008715:Methionine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D017382:Reactive Oxygen Species / Q000737:chemistry; D016339:Spectrometry, Mass, Fast Atom Bombardment; D013816:Thermodynamics; D014908:Triticum
chemical_list: D002147:Calmodulin; D010446:Peptide Fragments; D017382:Reactive Oxygen Species; D008715:Methionine; D006861:Hydrogen Peroxide; D000252:Calcium-Transporting ATPases; D002118:Calcium