An environmentally acceptable method for assaying the inhibition of .ALPHA.-amylase induction

Abstract

A facile a-amylase induction inhibition assay method was investigated. Induced a-amylase was determined using a microplate filled with a gel containing rice starch and Gellan gum. Embryoless barley seeds were incubated with GA 3 (10 Ϫ7 M) and appropriate ABA. Diluted incubation fluid was dispensed to the wells and the change of absorbance was measured. Induced a-amylase was calculated from the change of the absorbance. The results were almost equal to those of the Somogyi-Nelson method. As noxious wastewater containing heavy metal ions does not occur in this experiment, this method can be an environmentally acceptable assay method for ABA analogs. © Pesticide Science Society of JapanKeywords: a-amylase quantification, gibberellic acid, abscisic acid, a-amylase induction inhibition assay. IntroductionWhen barley seeds germinate, gibberellic acid (GA 3 ), first synthesized in the embryo, is transported to the aleurone layer and induces a-amylase biosynthesis in the aleurone layer. This process is specifically inhibited by plant hormone abscisic acid (ABA). 1) This inhibition of a-amylase biosynthesis occurs at the transcription stage and the inhibition of a-amylase mRNA synthesis is recognized as a specific activity of ABA.2) This inhibition of a-amylase induction is used as a specific bioassay method for ABA and its analogs.3) The Somogyi-Nelson method has been widely used to evaluate the amount of induced a-amylase by measuring the quantity of reducing sugars in the culture fluid. 4,5) Although the method has excellent sensitivity for reducing sugars, wastewater including arsenic and copper compounds arises in the experimental procedure. 6)In 1998, Satoyama et al. reported a convenient method for measuring a-amylase activity. The principle of this method is based on the turbidity change caused by the hydrolysis of precipitated starch by added a-amylase. 7,8) This method seems valid for our purpose since it enables us to deal with many samples automatically and to exclude the use of toxic metal salts. We tried to apply this method for the a-amylase induction assay. In this paper, we will discuss the improved a-amylase measuring method and its application in the research of ABA analogs. Materials and Methods Materials and reagentsGibberellic acid (GA 3 ) and (S)-abscisic acid were obtained from Kyowa Hakko Co. Ltd. and the barley seeds (Hordeum vulgare L. cv. Ichibanboshi) were from Kumamoto Prefectural Agricultural Research Center. All other reagents and solvents were from Wako Pure Chem. Industries (Osaka). Preparation of the substrate plate Preparation of the substrate plate solidified with agarThe substrate plate solidified with agar was prepared as follows: rice starch (1.5 g) and agar (1.5 g) were dissolved in 100 ml of citrate buffer (0.1 M, pH 6.0) in a boiling water bath until the suspension became a clear solution. The hot solution was dispensed into the wells of the microplate, which was left at room temperature until the solution solidified. The absorbance of each well at 620 nm was measured using a mi...

A High-Throughput Assay for Quantification of Starch Hydrolase Inhibition Based on Turbidity Measurement

“…Samples containing insoluble solids will interfere with the light transmittance, and the interference is related to the size, shape, and composition of the particles and to the wavelength of the incident light . Turbidity can be measured by using microplate UV–vis reader, which is mostly applied for monitoring cell growth dynamics. , The purpose of this study is to fully develop and validate a high-throughput method based on turbidity measurement for investigation of the dynamic of enzymatic hydrolysis of starch particles. We hope our method can be generally applied in research on novel starch hydrolase inhibitors from vastly diverse botanical sources, starch microstructure, and digestibility relationship and in formulations of foods with low glycemic index.…”