Freeze-etching study on membrane ultrastructural changes caused by freezing: cooling rate-dependent intramembrane particle aggregation in erythrocyte stripped ghosts.

The changes of membrane ultrastructures by freezing stresses were examined on stripped ghosts which were made by removing almost all peripheral membrane proteins from human erythrocyte membranes. By freezing these stripped ghost membranes showed cooling rate-dependent intramembrane particle (IMP) aggregation. With the cooling rates at and faster than 30,000 degrees C/min, their IMPs were evenly distributed on the fracture faces. However, cooling rates at and slower than 8000 degrees C/min resulted in IMP aggregation. The degree of IMP aggregation increased in parallel with decreasing cooling rates. Without freezing, the IMP aggregation in stripped ghosts could be induced by exposing these ghosts to hypertonic salt solutions, but lowering the temperature did not affect IMP aggregation. The cooling rate-dependent IMP aggregation during freezing was suppressed by adding cryoprotective agents which were known to reduce the salt concentration of the medium during freezing. It is suggested that the IMP aggregation in stripped ghosts by freezing occurs by exposure to concentrated salt solutions during freezing. This result indicates the possibility that IMP aggregation may arise during slow freezing of some biomembranes as a result of an increase in salt concentration rather than as a result of reduction in temperature.