Selective chemical modification of plasma membrane ectoenzymes

As part of an investigation of the organization of cell surface macromolecular assemblies, we have treated intact central nervous system cells with chemical probes which react convalently with proteins and aminophospholipids. Selective alterations of the enzymatic activities of ecto-ATPases, ecto-5'-nucleotidases and cholinesterases were obtained under appropriate reaction conditions. The cross-linking reagent, 1,5-difluoro-2,4-dinitrobenzene, was a potent inactivator of ecto-ATPase of C6 glioblastoma, IMR-32 neuroblastoma and of a primary rat astroblast cell line (RB). Ecto-5'-nucleotidase and acetylcholinesterase were less sensitive to difluorodinitrobenzene. 1-Fluoro-2,4-dinitrobenzene at concentrations which inactivated ecto-ATPase had little effect on ecto-5'-nucleotidase. Conversely, 2,4,6-trinitrobenzenesulfonic acid was a potent inactivator of ecto-5'-nucleotidase but had no effect on ecto-ATPase. The difluorodinitrobenzene inactivation of ecto-ATPase and of ecto-5'-nucleotidase as well as the fluorodinitrobenzene inactivation of ecto-ATPase could be prevented by the presence of the appropriate substrates in the reaction medium. In the presence of protecting nucleotide substrates, a decrease in reactivity with proteins and lipids was observed when the isotopic probe fluorodinitro[3H]-benzene was used.