Abstract

Dithiothreitol, a disulfide bridge-reducing agent, decreased 10-fold the apparent activation constant of (-)-isoproterenol for the adenylate cyclase system in C6 glioma cells, rat liver cells, and avian erythrocytes. In liver plasma membranes, the apparent activation constant of glucagon for the adenylate cyclase was unchanged. In C6 glioma cells, dithiothreitol also decreased by 10-fold the affinity of the beta adrenergic receptor for (-)-[³H] dihydroalprenolol without modifying the total number of sites. Thus, with a submaximal concentration of (-)-[³H]dihydroalprenolol (10 nM), the specific binding was reduced by 70-80% in the presence of dithiothreitol (10 mM) or other disulfide bridge-reducing agents: 2-mercaptoethanol (50 mM) and cysteine (20 mM). Addition of dithiothreitol to intact cells or to broken cell preparations had a similar effect. The fall in affinity was due to a 3-4-fold increase in the dissociation rate constant and a 2-3-fold decrease in the association rate constant. The reduced receptor was reoxidized by simple exposure to air. The opening of the disulfide bridge may have changed the receptor spatial configuration, since there were 10-fold and 25-fold losses in affinity for the (-)- and (+)-isoproterenol isomers, respectively.