JUNCTIONAL SITES OF ERYTHROCYTE SKELETAL PROTEINS ARE SPECIFIC TARGETS OF TERT-BUTYLHYDROPEROXIDE OXIDATIVE DAMAGE

The oxidative denaturation of the erythrocyte membrane, which is consi dered a major cause of the haemolytic process, was evaluated upon 'in vitro' oxidative stress with tert-butylhydroperoxide. Biochemical and ultrastructural analyses were performed to point out the effect of thi s substance on the skeletal network, which is mainly responsible for r ed cell shape and viability. Moreover, cell morphology was observed by scanning electron microscopy and membrane rigidity assessed by EPR me asurements. The most relevant features of the membrane denaturation we re, (i) lipid peroxidation, as assessed by malonildialdehyde productio n, (ii) spectrin and ankyrin degradation with simultaneous globin bind ing to the membrane, as evidenced by electrophoretic pattern of red ce ll ghosts. These phenomena were related to the drug concentration in t he incubation medium, and accompanied by depletion of intracellular re duced glutathione. The denaturation of protein components hindered the release of spectrin in a hypotonic extraction medium and could be onl y partially reversed by dithiothreitol. The extensive membrane protein and lipid degradation, at high drug concentration, was coherent with a marked increase of membrane order (membrane 'rigidity'). No clusteri ng of intramembrane proteins was shown by the transmission electron mi croscopy images. At the same time scanning electron microscopy demonst rated shrinking and disco-stomatocytic deformation of erythrocytes. Ul trastructural analysis of the membrane skeleton by fluorescence-labell ing of spectrin and actin, allowed to point out that exposure to t-BHP caused the marginalization of spectrin and the rearrangement of actin molecules with formation of micro aggregates, so that a detachment of actin from the spectrin network was suggested. In addition to the gen eralized damage of red cell membrane, tert-butylhydroperoxide was foun d to induce a specific alteration of the skeletal network at the horiz ontal junction sites involving spectrin, actin, and protein 4.1 and th us to modify the cytoskeletal assembly. This effect on the membrane sk eletal components was consistent with the hypothesis that oxidative st ress plays a key role in the haemolytic process.