The purpose of the study was to measure ATPase activity (Na+/K+ and Ca2+) in fresh vesicles and vesicles that had been thawed following freezing in one of three media (sucrose, trehalose, and glycerol) and by two different techniques (dry ice/methanol and liquid nitrogen). Vesicles frozen in either sucrose or trehalose using dry ice/methanol resulted in preservation of 93% and 81% Na+/K+ ATPase activity, respectively. Freezing in liquid nitrogen resulted in loss of 82% and 68% Na+/K+ ATPase activity for vesicles resuspended in trehalose and sucrose, respectively. ATP-dependent 45Ca2+ uptake into IOVs was determined via rapid filtration. Optimal uptake activity was preserved (92%) when vesicles in sucrose were frozen in methanol/dry ice. Rapidly freezing these vesicles in liquid nitrogen decreased activity by 25%. Vesicles resuspended in trehalose lost 50% of their ATP-dependent calcium uptake when frozen either in methanol/dry ice or liquid nitrogen. Functional characterization of both Na+/K+ and Ca2+ ATPase activity suggests that the optimal method for freezing vesicles is to resuspend in sucrose and freeze in methanol/dry ice. Attempts to preserve in glycerol proved unsuccessful. Washing to remove the glycerol significantly decreased protein recovery and associated enzyme activity.
|Original language||English (US)|
|State||Published - Mar 20 1998|
ASJC Scopus subject areas
- Molecular Biology