sepharose and dimethyl-pimelimidate

Immobilization of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) from spinach leaves is described. This enzyme enables the fixation of carbon dioxide on a five-carbon sugar D-ribulose-1,5-bisphosphate (RuBP). Two different immobilization methods were employed: dicyclohexylcarbodiimide coupling on nylon membrane matrix and dimethylpimelimidate immobilization on protein A agarose. The reusability of immobilized enzymes, coupling efficiency, and temperature-activity relationship of soluble and immobilized Rubisco are presented. The immobilization imparted greater thermal and storage stability. The thermal deactivation rates of the immobilized enzymes were considerably lower than those of the soluble enzyme.

Topics: Carbon Dioxide; Dicyclohexylcarbodiimide; Drug Storage; Enzyme Activation; Enzyme Stability; Enzymes, Immobilized; Hydrogen-Ion Concentration; Imidoesters; Membranes, Artificial; Nylons; Plant Leaves; Ribulose-Bisphosphate Carboxylase; Sensitivity and Specificity; Sepharose; Spinacia oleracea; Temperature

Monoclonal antibodies reactive with defined T lymphocyte surface antigens were covalently coupled to protein A-Sepharose beads using the bifunctional imidoester, dimethyl pimelimidate. Sepharose-immobilized antibody reactive with T3 induced the proliferation of resting T lymphocytes in the presence of either recombinant interleukin 2 or phorbol myristate acetate. When monoclonal antibodies reactive with T3 and T4 were coupled to the same Sepharose bead (hereafter designated Sepharose (T3:T4)), proliferation was enhanced an average of three-fold. Similarly prepared Sepharose beads coupled to anti-T3 and anti-T8 also enhanced proliferation over that observed with anti-T3 alone. Sepharose (T3:T4) similarly increased the proliferation of T4+ lymphocytes and a T4+ clone but failed to enhance the proliferation of T8+ lymphocytes. The increased proliferation of T4+ lymphocytes resulted from a preferential activation of the T4+2H4- helper population over the T4+2H4+ suppressor-inducer population. The enhanced proliferation induced by Sepharose (T3:T4) could be completely inhibited by soluble anti-T4. These results suggest that perturbation of T3 may be a minimal signal for T cell activation and that the assembly of a multimeric complex including T3 and T4 may be required for optimal T cell activation.

Topics: Antibodies, Monoclonal; Antigens, Differentiation, T-Lymphocyte; Antigens, Surface; Cell Division; Cells, Cultured; Cross-Linking Reagents; Humans; Imidoesters; Lymphocyte Activation; Microspheres; Sepharose; T-Lymphocytes