The Accumulation Kinetics of The Small and Large Subunits of Ribulose 1,5-Bisphosphate Carboxylase During Chloroplast Development in Euglena Gracilis

359 p.Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1983.The Calvin cycle enzyme ribulose 1,5-bisophosphate carboxylase (RuBPCase) is composed of two subunits, a large subunit which is encoded and synthesized in the chloroplast, and a small subunit which is encoded in the nucleus and synthesized in the cytoplasm. By determining the accumulation kinetics of the two subunits during chloroplast development, we have initiated an investigation into intracellular regulation of protein-synthesis.RuBPCase was purified to homogeneity, and the subunits, separated by preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, injected into rabbits. The antibodies (IgG fractions) raised against the RuBPCase subunits were used in three different immunochemical techniques to determine the accumulation kinetics for both subunits during the light-induced chloroplast development of Euglena gracilis. The immunochemical techniques used were: indirect radioimmunoassay; direct radioimmunoassay; and radial radioimmunodiffusion, a novel technqiue, developed during the course of these experiments. All of the techniques were modified to include SDS in the immunochemical reactions because the large subunit is insoluble in the absence of SDS.Results of the immunochemical assays demonstrated that in Euglena the synthesis of S subunit begins immediately upon exposure of the cells to light, while the synthesis of L subunit begins only after a 5-hour lag period. Even at the end of a 72-hour greening period the molar concentration of S subunit in the cells is approximately 3x that of L subunit.U of I OnlyRestricted to the U of I community idenfinitely during batch ingest of legacy ETD