LIPID-PEROXIDATION AS THE MECHANISM OF MODIFICATION OF BRAIN 5'-NUCLEOTIDASE ACTIVITY INVITRO

The effect of lipid peroxidation on the Mg2+-independent and Mg2+-dependent activity of brain cell membrane 5′-nucleotidase was determined and the affinity of the active sites of Mg2+-dependent enzyme for 5′-AMP (substrate) and Mg2+ (activator) was examined. Brain cell membranes were peroxidized at 37°C in the presence of 100 μM ascorbate and 25 μM FeCl2 (resultant) for 10 min. The activity of 5′-nucleotidase and lipid peroxidation products (thiobarbituric acid reactive substances) were determined. At 10 min, the level of lipid peroxidation products increased from 0.20±0.10 to 17.5±1.5 nmoles malonaldehyde/mg membrane protein. The activity of Mg2+-independent 5′-nucleotidase increased from 0.201±0.020 in controls to 0.305±0.028 μmol Pi/mg protein/hr in peroxidized membranes. In the presence of 10mM Mg2+, the activity increased by 5.8-fold in the peroxidized membrane preparation in comparison to 14-fold in control In peroxidized preparation, the affinity of active site of Mg2+-dependent 5′-nucleotidase for 5′-AMP tripled, as indicated by a significant decrease in Km (Km=95±2 μM AMP for control;Km=32±2 μMAMP for peroxidized). Vmax was significantly reduced from 3.35±0.16 in control to 1.70±.09 μmoles Pi/mg protein in peroxidized membranes. The affinity of the active site for Mg2+ significantly increased (Km=6.17±0.37 mM Mg2+ for control;Km=4.0±0.31 peroxidized). The data demonstrate that lipid peroxidation modifies the Mg2+-dependent 5′-nucleotidase function by altering the active sites for both the substrate and the activator. The modification of the 5′-nucleotidase activity and the loss of Mg2+-dependent activation observed in this in-vitro study are similar to the changes previously observed by us in the hypoxic brain in-vivo. This suggests that lipid peroxidation which specifically alters the active site may be the underlying mechanism of the modification of 5′-nucleotidase during hypoxia. © 1990 Plenum Publishing Corporation.