Incorporation of monoclonal antibodies into cells by osmotic permeabilization. Effect on cellular metabolism.
Incorporation of asparagine synthetase-specific monoclonal antibodies into L5178Y D10/R (L-asparaginase-resistant) murine lymphoma cells by osmotic lysis of pinocytic vesicles was used to evaluate the potential of the technique for macromolecular incorporation for metabolic studies. Nonspecific effects of the incorporation procedure included temporary inhibition of protein and DNA synthesis by 80-85% and a transitory loss of membrane integrity. Cells incorporated with an antibody inhibitory to tumor cell asparagine synthetase showed increased dependence upon an exogenous source of asparagine in the culture medium, while cells incorporated with a control antibody were not affected. These studies demonstrated that incorporation of inhibitory monoclonal antibodies into cells can be used to study the short term metabolic role of specific enzymes; however, the metabolic effects induced by the specific macromolecule must be evaluated within the context of the nonspecific effects caused by the osmotic treatment required for incorporation.
Chakrabarti
R
R
Department of Chemistry, University of Nebraska, Lincoln 68588-0304.
Pfeiffer
N E
NE
Wylie
D E
DE
Schuster
S M
SM
D000911:Antibodies, Monoclonal; D001217:Aspartate-Ammonia Ligase / Q000276:immunology*; D015153:Blotting, Western; D002462:Cell Membrane / Q000648:ultrastructure; D002463:Cell Membrane Permeability; D003593:Cytoplasm / Q000378:metabolism / Q000648:ultrastructure; D004247:DNA / Q000096:biosynthesis; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008025:Ligases / Q000276:immunology*; D008223:Lymphoma / Q000378:metabolism* / Q000648:ultrastructure; D008854:Microscopy, Electron; D009994:Osmolar Concentration; D009997:Osmotic Pressure; D010873:Pinocytosis; D014176:Protein Biosynthesis; D013861:Thiocyanates; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D004247:DNA; D007770:L-Lactate Dehydrogenase; D008025:Ligases; D001217:Aspartate-Ammonia Ligase; D016650:Fluorescein-5-isothiocyanate