A modified amylase assay, using a fluorescent substrate, and its application to a study of the rat parotid gland in vitro.
A modified fluorescence assay for alpha-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 alpha-amylase is incubated with the substrate for 5 min at 30 degree C in a final volume of 750 microliter. 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 alpha-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 alpha-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.
D000681:Amylases / Q000032:analysis*; D000687:Amylopectin; D000818:Animals; D066298:In Vitro Techniques; D007545:Isoproterenol / Q000494:pharmacology; D008297:Male; D010306:Parotid Gland / Q000187:drug effects / Q000201:enzymology*; D051381:Rats; D013050:Spectrometry, Fluorescence; D013373:Substance P / Q000031:analogs & derivatives / Q000494:pharmacology; D000516:alpha-Amylases / Q000032:analysis* / Q000378:metabolism
D013373:Substance P; C033940:substance P, amino(4)-Phe(7)-; D000687:Amylopectin; D000681:Amylases; D000516:alpha-Amylases; D007545:Isoproterenol