Validation of assay for measuring acetyl-coenzyme a carboxylase activity in grasses using malachite green

Abstract

Acetyl-CoA carboxylase (ACCase) is one of the most important enzymes as a herbicide target in gramineous plant species, however, assay methods for the enzyme are primarily limited to those using radioisotopes (RI). Typically, the measurement method that uses RI necessitates specialized facilities and equipment, and involves complex procedures throughout the experiment. As another method for detecting ACCase activity, the colorimetric method using malachite green (MG) is also known. However, reports on this method are limited, and information regarding the simplicity of the procedure and the scope of its application remains unclear. To better understand the method using MG and to develop a simpler assay method, crude enzymes extracted from various target-site resistant (TSR) biotypes of blackgrass (Alopecurus myosuroides) were examined in enzyme inhibition tests. As a result, this method was able to accurately detect the relationship between the chemical classes of ACC inhibitors and cross-resistance to specific TSRs. Moreover, the ACCase activity of other grass species was also examined using this method. By using crude enzymes from various species and a commercially available phosphatase kit containing MG, ACCase activity was detectable with good accuracy. In addition, enzyme inhibition studies using ACCase inhibiting herbicides revealed that this method reproduced results similar to those obtained with the RI method. The Z′-factor, an index of high-throughput screening (HTS), was around 0.7, indicating that it is an excellent screening system. These results suggest that the assay method using MG is very simple, labor-saving, and accurate with a throughput much higher than that of the existing RI method. Therefore, it is strongly suggested that the method could replace the RI method in most cases. These results indicate that it is applicable to HTS for ACCase inhibitors.