Fig. 8

Inhibition and cation dependence of TBI-induced endonuclease activity. (A) The nuclease activity in cytosolic extracts (0.5 mg/ml final concentration) from injured rat cortex isolated 72 hr after TBI was assayed in the presence or in the absence of 20 • M aurintricarboxylic acid (ATA), 10 mM EDTA, 2 mM ZnSO₄, or the protein extracts were preheated at 70°C for 15 min before the reaction. The reactions were carried for 1 hr at 37°C with 2 • g of pCR2.1 plasmid DNA in the buffer consisting of 10 mM Hepes (pH7.4), 50 mM KCl, 3 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 3 mM ATP, 10 mM phosphocreatine, 50 • g/ml creatine kinase, and 20% glycerol. The DNA was then analyzed by electrophoresis through a 1.5% agarose gel and ethidium bromide staining. (B) Cation dependence of TBI-induced endonuclease activity was assayed in the presence of either 5 mM MgCl₂, 5 mM CaCl₂, or 2 mM ZnSO₄. The reactions were carried and DNA was analyzed as described for (A). This results show that the endonuclease activity induced in rat brain cortex following TBI depends on the presence of Mg²⁺ and Ca²⁺, but is not inhibited by Zn²⁺. Data are representative of three experiments with similar results.