Sampedro J G, Guerra G, Pardo J P, Uribe S
Departamento de Bioquímica, Universidad Nacional Autónoma de México, México D.F., 04510, México.
Cryobiology. 1998 Sep;37(2):131-8. doi: 10.1006/cryo.1998.2109.
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.
在冷冻干燥和复水过程中，通过增加碳水化合物的浓度，可以保留乳酸克鲁维酵母质膜中H⁺-ATP酶的活性。当H⁺-ATP酶在没有碳水化合物的情况下进行冷冻干燥时，活性丧失。碳水化合物的保护效率如下：海藻糖＞麦芽糖＞蔗糖＞葡萄糖＞半乳糖。每种碳水化合物在浓度为每毫克蛋白质20毫克碳水化合物或更高时表现出最大保护作用。通过内源荧光测量未检测到复水后的H⁺-ATP酶有结构变化。在冷冻干燥和复水过程中，每毫克蛋白质20毫克的海藻糖能完全保护酶活性。复水温度至关重要；在20℃或更低温度下，活性可完全保留，而在30℃、40℃或50℃时，活性随温度成比例下降。用海藻糖保护的冷冻干燥H⁺-ATP酶在不同温度下储存长达60天。在4℃储存可保留大部分酶活性，而在20℃或30℃储存则导致活性丧失。海藻糖对H⁺-ATP酶的保护作用表明，这种碳水化合物可能在处理过程中保护其他膜酶不被失活。
