Involvement of amino-acid side chains of membrane proteins in the binding of glutathione to pig cerebral cortical membranes.
Glutathione (GSH), a general antioxidant and detoxifying compound, is the most abundant thiol-containing peptide in the central nervous system. It has been earlier shown to regulate the functions of glutamate receptors and to possess specific binding sites in both neurons and glial cells. The possible involvement of disulfide bonds, cysteinyl, arginyl, lysyl, glutamyl, and aspartyl residues in the binding of tritiated GSH to specific sites in pig cerebral cortical synaptic membranes was now studied after covalent modification of membrane proteins. Treatment of synaptic membranes with the thiol-modifying reagents 5,5'-dithio-bis(2-nitrobenzoate) (DTNB) and 4,4'-dithiodipyridine (DDP) dramatically enhanced the binding of [3H]GSH in a dose-dependent manner. Dithiothreitol (DTT) alone reduced the binding, but pretreatment of the membranes with DTT potentiated the enhancing effect of DTNB. On the other hand, when the modification with DTNB was followed by treatment with DTT, the enhancement by DTNB was completely reversed. N-ethylmaleimide, a thiol alkylating agent, and phenylisothiocyanate, a thiol- and amino-group modifying compound, reduced the binding, and their effects were additive. The guanidino-modifying agent phenylglyoxal reduced the binding but the carboxyl-modifying reagent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide had no significant effect. The results indicate that cysteinyl side chains and disulfide bonds are essential in the binding of GSH to membrane proteins and that arginyl and lysyl side chains may also be directly involved in this process.
Hermann
András
A
Tampere Brain Research Center, Medical School, University of Tampere, Finland.
Varga
Vince
V
Oja
Simo S
SS
Saransaari
Pirjo
P
Janáky
Réka
R
D000596:Amino Acids / Q000502:physiology*; D000818:Animals; D002540:Cerebral Cortex / Q000378:metabolism*; D004220:Disulfides / Q000494:pharmacology; D004228:Dithionitrobenzoic Acid / Q000494:pharmacology; D005978:Glutathione / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*; D011725:Pyridines / Q000494:pharmacology; D013552:Swine; D013570:Synaptic Membranes / Q000187:drug effects / Q000378:metabolism*
D000596:Amino Acids; D004220:Disulfides; D008565:Membrane Proteins; D011725:Pyridines; C007542:4,4'-dipyridyl disulfide; D004228:Dithionitrobenzoic Acid; D005978:Glutathione