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Superoxide dismutase within the bovine erythrocyte membrane

1981; Elsevier BV; Volume: 645; Issue: 1 Linguagem: Inglês

10.1016/0005-2736(81)90513-7

1879-2642

A. Petkau, T.P. Copps, K. Kelly,

Mass Spectrometry Techniques and Applications

(1) Membrane associated superoxide dismutase molecules in intact bovine erythrocytes were not readily accesible for reaction with rabbit 125I-labelled antibodies, specific for the enzyme. Membrane alterations in ghost formation resulted in a 17-fold increase in uptake of 125I-labelled antibody. The uptake of 125I-labelled antibody by inside-out vesicles and intact ghost was 3 : 1 in favour of the cytoplasmic surface, indicating an asymmetric distribution across the cell membrane. (2) Assay for enzyme activity after polyacrylamide gel electrophoresis of chloroform-ethanol extracts of two-day-old ghosts indicated the presence of 320 ± 105 mol of active enzyme per ghosts or 0.20 ± 0.07% of the cellular content. When examined over a 30-day storage period in-situ, the enzyme activity decreased by apparent first order kinetics to an equivalent of 110 ± 33 mol per ghost. The amount of protein extracted with chloroform-ethanol from ghost and which electrophoresed with the same mobility as superoxide dismutase was in a large excess over enzyme protein, calculated from the measured activity, and increased with time of storage. Radioimmunoassay, however, detected no change in enzyme protein with storage. (3) The non-ionic detergent, Nonidet P-40, extracted twice as much active enzyme from ghosts as chloroformethanol. The detergent-extracted enzyme with the mobility of superoxide dismutase was resolved, using an immunosorbent of rabbit anti-superoxide dismutase antibody coupled to Sepharose, into an active component which bound to the immunosorbent and an inactive fraction, 0.23% of which could be restored to active superoxide dismutase by reconstitution with copper. The reactivated enzyme (2570 ng) was equal to 123% of the amount (2097 ng) detected by radioimmunoassay in the original membrane extract. However, this unique membrane component, whether in its active or reactivated form, could not be detected by the radioimmunoassay which employed antibodies directed against cytoplasmic superoxide dismutase.