Superoxide modification and inactivation of a neuronal receptor-like complex.
Excessive superoxide (O(-)(2)) formation is toxic to cells and organisms. O(-)(2) reacts with either iron-sulfur centers or cysteines (Cys) of cytoplasmic proteins. Reactions with membrane proteins, however, have not been fully characterized. In the present studies, the reaction of O(-)(2) with a protein complex that has glutamate/N-methyl-D-aspartate (NMDA) receptor characteristics and with one of the subunits of this complex was examined. Exposure of the complex purified from neuronal membranes and the recombinant glutamate-binding protein (GBP) subunit of this complex to the O(-)(2)-generating system of xanthine (X) plus xanthine oxidase (XO) caused strong inhibition of L-[3H]glutamate binding. Inhibition of glutamate binding to the complex and GBP by O(-)(2) was greater than that produced by H(2)O(2), another product of the X plus XO reaction. Mutation of two cysteine (Cys) residues in recombinant GBP (Cys(190,191)) eliminated the effect of O(-)(2) on L-[3H]glutamate binding. Both S-thiolation reaction of GBP in synaptic membranes with [35S]cystine and reaction of Cys residues in GBP with [3H]NEM were significantly decreased after exposure of membranes to O(-)(2). Inhibition of cysteylation of membrane GBP by O(-)(2) was still observed after iron chelation by desferrioxamine, albeit diminished, and was not altered by the presence of catalase. Overall, the results indicated that GBP exposure to O(-)(2) modified Cys residues in this protein. The modification was not characterized but it was probably that of disulfide formation.
Agbas
A
A
Department of Pharmacology and Toxicology, University of Kansas, Lawrence, KS 66047, USA.
Chen
X
X
Hong
O
O
Kumar
K N
KN
Michaelis
E K
EK
D000818:Animals; D003545:Cysteine / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000378:metabolism; D018698:Glutamic Acid / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D016202:N-Methylaspartate / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding / Q000187:drug effects; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology; D016194:Receptors, N-Methyl-D-Aspartate / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism / Q000494:pharmacology*; D013570:Synaptic Membranes / Q000737:chemistry; D014316:Tritium; D019820:Xanthine / Q000378:metabolism; D014969:Xanthine Oxidase / Q000378:metabolism
D008565:Membrane Proteins; D017382:Reactive Oxygen Species; D016194:Receptors, N-Methyl-D-Aspartate; D014316:Tritium; D013481:Superoxides; D019820:Xanthine; D018698:Glutamic Acid; D016202:N-Methylaspartate; D014969:Xanthine Oxidase; D003545:Cysteine; D005033:Ethylmaleimide