Mechanism of augmentation of the antibody response in vitro by 2-mercaptoethanol in murine lymphocytes : II. A major role of the mixed disulfide between 2-mercaptoethanol and cysteine

Abstract

Five thiol compounds including 2-mercaptoethanol (2-ME) were examined for their augmenting effects on in vitro antibody response to sheep erythrocytes. Three compounds were effective with the following order of activity; 2-ME > dithiothreitol > cysteamine. Glutathione and thioglycollate failed to enhance the response. The same order of effectiveness was seen in the stimulation of [ 35S]cystine uptake by murine lymphocytes by these thiols. Murine lymphocytes took up cysteine five to six times more rapidly than cystine. It is, however, unlikely that 2-ME stimulation of cystine uptake is solely due to the reduction of cystine into cysteine, because 2-ME was still stimulatory after free thiol groups had disappeared in the medium containing 2-ME and [ 35S]cystine. The mixed disulfide of cysteine with 2-ME (Cys-2-ME) was found to be an only product after free thiols had been oxidized. [ 35S]Cys-2-ME was taken up by the lymphocytes with a comparable rate to cysteine via a transport system common to that of leucine and phenylalanine. Cysteine was, however, transported via a different route. It was observed that Cys-2-ME was readily metabolized to cysteine and glutathione after the uptake. Cys-2-ME added to cystine-free RPMI 1640 medium could support the antibody response as efficiently as cystine plus 2-ME. These observations strongly suggest that 2-ME stimulates cystine uptake and, therefore, enhances the antibody response through the formation of the mixed disulfide with cysteine.