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

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.