Complement lysis of nucleated cells: Effect of temperature and puromycin on the number of channels required for cytolysis☆

Abstract We have previously shown that lysis of a nucleated mammalian cell requires several complement channels unlike lysis of erythrocytes and that this difference is due primarily to rapid elimination of channels from the plasma membrane. We have now investigated this problem further by studying the rate of channel elimination at low temp, the osmotic fragility of the cells, and the effectiveness of the membrane-associated ion pumps. When complement channels were formed for 3 min at 37°C, followed by prolonged incubation at 2°C, the C6 lytic dose-response curves indicated that a single channel was required for lysis of a cell, whereas multiple channels were required when the entire process was carried out at 30°C. The shift from multi- to one-hit lytic behavior can be explained by the drastic reduction in the rate of channel elimination at low temp. C6 lytic dose-response curves with puromycin-treated cells were also found to display one-hit behavior, but, in this case, the rate of channel elimination was reduced only about 35–40% (which would not suffice to explain the one-hit lytic characteristics). However, cell death was more extensive for puromycin-treated cells than normal cells after incubation in buffers of low ionic strength, suggesting that an increase in osmotic fragility may be a contributing factor in the shift from multi- to one-hit behavior. Blocking of the membrane-associated Na + /K + -ATPases with ouabain did not affect the multi-channel requirement. Presumably, this means that the ion pumping rate does not significantly influence the number of channels required for lysis.