Modification of Plasma Membrane DT-Diaphorase Activity Upon Transformation: A Comparison between Astrocytes in Primary Culture and C6 Glioblastoma Cells

Abstract

Acceptor oxidoreductase activity using NADH or NADPH as electron donnors, dichloroindophenol (DCIP) and potassium ferricyanide [K₃ Fe(CN)₆] as external electron acceptors and rotenone, dicoumarol and antimycin as inhibitors, was investigated in plasma membrane isolated from neonatal rat astrocyte primary culture and the plasma membrane of glioblastoma cell lines (C6). Astrocytic plasma membranes were enriched approximately 10 fold in both NADH and NADPH-DCIP oxidoreductase specific activities (300 nmol min/mg protein), whereas plasma membranes derived from C6 cells possessed NADH and NADPH-ferricyanide oxidoreductase activity (9 nmol ming/mg protein) — typical of DT-diaphorase system, but no enrichment of those activities was observed in the isolated C6 plasma membrane. NADPH-DCIP reductase activity of astrocyte plasma membranes was sensitive towards dicoumarol and antimycin (45% and 71% inhibition, respectively). The NADH or NADPH-ferricyanide reductase activity of C6 plasma membranes was inhibited by rotenone, dicoumarol and antimycin (40%, 60% and 10%, respectively). Both cell types displayed NADH-DCIP cell surface reductase activity. In addition, NADPH-DCIP reductase activity was found at the surface of C6 cells. Astrocyte cell surface activity was sensitive towards dicoumarol exclusively (70% inhibition) whereas in the case of C6 cells, rotenone, dicoumarol or antimycin inhibited the enzymatic activity.