通过DNA酶I抑制试验评估在各种条件下处理的肌动蛋白-肌球蛋白复合物中肌动蛋白的变性情况。

Estimation of denaturation of actin in the actin-myosin complex treated with various conditions by DNAase I inhibition assay.

Ikeuchi Y, Iwamura K, Suzuki A

Laboratory of Biochemistry and Technology of Animal Products, Faculty of Agriculture, University of Niigata, Japan.

Int J Biochem. 1991;23(10):985-9. doi: 10.1016/0020-711x(91)90134-9.

Abstract

Experiments were conducted to evaluate whether DNAase I (EC 3.1.4.5) inhibition assay was a valuable tool to study the denaturation of actin in the actin-myosin complex treated with various conditions. 2. A sample containing F-actin or natural actomyosin(myosin B) was treated with KI-ATP solution to convert a form which inhibits DNAase I as effectively as G-actin, and the total amount of native actin was determined by DNAase I inhibition assay. 3. On the basis of the values for remaining native actin in the sample obtained by this assay, a percentage of denaturation of actin during treatment was calculated. 4. The present result demonstrated that DNAase I inhibition assay was easy to perform, very sensitive (0.5-2.0 microgram actin) and highly specific for estimating denaturation of actin in the actin-myosin complex treated with heat or high salt concentrations. 5. In addition, the use of DNAase I and standard G-actin preparations stored frozen at -80 degrees C for the assay was found to be possible within a fixed period of time (about 2 weeks), which was helpful in monitoring the denaturation process of actin treated under various conditions for a long period.

摘要

进行实验以评估脱氧核糖核酸酶I（EC 3.1.4.5）抑制试验是否是研究在各种条件下处理的肌动蛋白-肌球蛋白复合物中肌动蛋白变性的有价值工具。2. 用碘化钾-三磷酸腺苷溶液处理含有F-肌动蛋白或天然肌动球蛋白（肌球蛋白B）的样品，以转化为一种与G-肌动蛋白一样有效抑制脱氧核糖核酸酶I的形式，并通过脱氧核糖核酸酶I抑制试验测定天然肌动蛋白的总量。3. 根据通过该试验获得的样品中剩余天然肌动蛋白的值，计算处理过程中肌动蛋白的变性百分比。4. 目前的结果表明，脱氧核糖核酸酶I抑制试验易于操作，非常灵敏（0.5-2.0微克肌动蛋白），并且对于估计在加热或高盐浓度处理的肌动蛋白-肌球蛋白复合物中肌动蛋白的变性具有高度特异性。5. 此外，发现使用脱氧核糖核酸酶I和保存在-80℃冷冻的标准G-肌动蛋白制剂进行该试验在固定时间段（约2周）内是可行的，这有助于长期监测在各种条件下处理的肌动蛋白的变性过程。