Superoxide modification and inactivation of a neuronal receptor-like complex

Abstract

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 2O 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.