Citation

This study investigated two independent aspects of lipopolysaccharide (LPS) induced murine B cell mitogenic responses. First, two-dimensional gels of lysates from LPS-stimulated B lymphocytes were compared with two-dimensional gels of lysates from control nonstimulated B lymphocytes. Densitométrie readings of the protein spots on these gels were determined. No difference was found qualitatively or quantitatively between the proteins of cell lysates from stimulated and non-stimulated cell cultures after 24 hours of incubation. Although these results were unexpected, it was felt to be a valid estimation of protein synthetic events in the cell. It is possible that other types of molecular events, changes in membrane potential, calcium influx or changes in cyclic nucleotides, may be more important in the first 24 hours or LPS stimulation. In addition, it was found that the general protein content of the cell does not vary significantly (less than 6%) between T lymphocytes and macrophages, and B lymphocytes which suggests that the basic protein content of these cell types was identical. Second, lipid A binding proteins were identified from spleen cell lysates. Lipid A is the portion of LPS which is responsible for most of the biological activity of that molecule. The proteins which were identified were 62,000, 60,000 and 28,000 daltons. These proteins could be identified in cell lysates from both LPS responsive and hyporesponsive mouse strains. The 62,000, 60,000 and 28,000 dalton proteins bind lipid A with a high affinity, such that the lipid Aprotein complex was maintained even in a sodium dodecyl sulfatepolyacrylamide (SDS-polyacrylamide) gel. A fourth protein which had a molecular weight of 45,000 daltons was also found in the sucrose gradient fraction which contained increased amounts of 1251 lipid A, the radioactive tracer in this system. This molecule failed to bind 1^1 lipid A in an SDS-polyacrylamide gel which indicated that it either binds 125i lipid A but is dissociated by SDS or that a second nondetectable protein binds the 125i lipid A. The fact that the 60,000 and 62,000 dalton protein could be co-isolated with the 45,000 dalton protein raises the possibility that these two molecules may be aggregated by LPS on cell surface.