A modified amylase assay, using a fluorescent substrate, and its application to a study of the rat parotid gland in vitro

Abstract

A modified fluorescence assay for α-amylase activity is described. The method employs amylopectin anthranilate as substrate and offers the advantages of economy of time and resources over a previously described technique using the same substrate. The sample containing α-amylase is incubated with the substrate for 5 min at 30°C in a final volume of 750 μl. The fluorescent products of the reaction are separated from the substrate by the addition of methanol, and the methanol-soluble fluorescence is measured in a fluorescence spectrometer. A highly reproducible linear relationship between fluorescence and α-amylase activity is obtained for enzyme activities up to 2 units. The absolute sensitivity of the assay under these conditions was estimated to be 0.02 EU (≡0.08 EU ml −1). The application of the assay method to a study of the effects of isoprenaline and substance P-like peptides on the release of α-amylase from rat parotid gland slices is described. The assay is particularly suitable for studies on agonists, such as substance P, which have a low ceiling effect in terms of amylase release.