Trehalose-Mediated Protection of the Plasma Membrane H -ATPase from Kluyveromyces lactis during Freeze-Drying and Rehydration Article uri icon

abstract

  • During freeze-drying and rehydration, the activity of the H -ATPase from the plasma membrane of Kluyveromyces lactis was preserved by increasing concentrations of carbohydrates. When the H -ATPase was freeze-dried in the absence of carbohydrates the activity was lost. The protective efficiency of carbohydrates was as follows: trehalose > maltose > sucrose > glucose > galactose. Each carbohydrate exhibited the maximal protection at a concentration of 20 mg carbohydrate per milligram of protein or above. No structural changes of the rehydrated H -ATPase were detected by intrinsic fluorescence measurements. Trehalose, at 20 mg/mg protein, protected the enzyme activity completely during freeze-drying and rehydration. Rehydration temperature was critical; at 20°C or below, activity was fully retained, while at 30, 40, or 50°C activity decreased in proportion with temperature. The trehalose-protected freeze-dried H -ATPase was stored at different temperatures for up to 60 days. Storage at 4°C resulted in retention of most of the enzymatic activity, while storage at 20 or 30°C resulted in loss of activity. The protection of the H -ATPase by trehalose suggests that this carbohydrate might protect other membrane enzymes from inactivation during handling. © 1998 Academic Press.
  • During freeze-drying and rehydration, the activity of the H%2b-ATPase from the plasma membrane of Kluyveromyces lactis was preserved by increasing concentrations of carbohydrates. When the H%2b-ATPase was freeze-dried in the absence of carbohydrates the activity was lost. The protective efficiency of carbohydrates was as follows: trehalose > maltose > sucrose > glucose > galactose. Each carbohydrate exhibited the maximal protection at a concentration of 20 mg carbohydrate per milligram of protein or above. No structural changes of the rehydrated H%2b-ATPase were detected by intrinsic fluorescence measurements. Trehalose, at 20 mg/mg protein, protected the enzyme activity completely during freeze-drying and rehydration. Rehydration temperature was critical; at 20°C or below, activity was fully retained, while at 30, 40, or 50°C activity decreased in proportion with temperature. The trehalose-protected freeze-dried H%2b-ATPase was stored at different temperatures for up to 60 days. Storage at 4°C resulted in retention of most of the enzymatic activity, while storage at 20 or 30°C resulted in loss of activity. The protection of the H%2b-ATPase by trehalose suggests that this carbohydrate might protect other membrane enzymes from inactivation during handling. © 1998 Academic Press.

publication date

  • 1998-01-01