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

Abstract

1. 1. 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. 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. 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. 4. The present result demonstrated that DNAase I inhibition assay was easy to perform, very sensitive (0.5–2.0 μg actin) and highly specific for estimating denaturation of actin in the actin-myosin complex treated with heat or high salt concentrations. 5. 5. In addition, the use of DNAase I and standard G-actin preparations stored frozen at −80°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.