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 degrees C or below, activity was fully retained, while at 30, 40, or 50 degrees 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 degrees C resulted in retention of most of the enzymatic activity, while storage at 20 or 30 degrees 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.
Departamento de Bioquímica, Universidad Nacional Autónoma de México, México D.F., 04510, México.
J G
JG
Sampedro
G
G
Guerra
J P
JP
Pardo
S
S
Uribe
D002462:Cell Membrane; D003451:Cryoprotective Agents / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D005612:Freeze Drying; D007716:Kluyveromyces; D006180:Proton-Translocating ATPases; D014199:Trehalose / Q000494:pharmacology*
D003451:Cryoprotective Agents; D014199:Trehalose; D006180:Proton-Translocating ATPases