Freeze-thaw damage in isolated lobster sarcoplasmic reticulum membranes: A model system for membrane damage

Sarcoplasmic reticulum membrane vesicles (SRV), isolated from the abdominal muscle of Maine lobsters, were put through a freeze-thaw cycle in order to study membrane freezing damage on a molecular basis, The major membrane protein in SRV is a (Ca²⁺ − Mg²⁺) ATPase capable of accumulating Ca²⁺ with the concomitant hydrolysis of ATP, After being frozen and thawed in the presence of NaCl, the SRV showed an increased ATPase activity and a decreased ability to accumulate Ca²⁺. The degree of increased ATPase activity and decreased Ca²⁺ accumulation was dependent upon the NaCl concentration (damage increased with increased NaCl concentration) and cooling rate (damage was only observed at slow cooling rates, i.e., less than 10 °C/min). Slow thawing rates also increased the amount of damage.The freeze-thaw damage of the SRV membranes is probably not due to osmotic shock, since the vesicles are quite resistant to osmotic stress and are highly permeable to small molecules and monovalent ions. Incubation of the SRV in 2 NaCl at 22 °C has no effect on Ca²⁺ accumulation whereas freezing in 0.25 NaCl totally abolishes their ability to take up Ca²⁺. Thus, a combination of salt and low temperature is necessary for damage. The freeze-thaw damage can be largely prevented by the addition of DMSO, glycerol, or PVP. The factors above have implications for the storage of tissue or membranes for subsequent analysis of membrane-bound enzymes. The SRV mimic the behavior of cells in their response to cooling and thawing rates, salts and cryoprotectants.