Microplate reader-based kinetic determination of alpha-amylase activity: application to quantitation of secretion from rat parotid acini.
A coupled enzyme assay for measuring alpha-amylase activity was adapted for analysis with a microplate reader. Activity was quantified by monitoring the cleavage of p-nitrophenol from a chemically defined substrate at 405 nm. Features of this assay method include: low sample volume (10 microliters); economical use of reagent (200 microliters); increased precision due to kinetic nature of assay; linearity with amylase content to 2600 U/liter; capability of processing up to 96 samples within 10 min; facilitated data analysis using readily available software. The ability to rapidly measure amylase content of a large number of samples permitted sophisticated analysis of alpha-amylase secretion patterns from dispersed rat parotid acinar cells. Examination of the dose-dependent increase in the rate of amylase secretion stimulated by carbachol revealed a biphasic response at higher concentrations, where a rapid and transient increase in amylase release was consistently observed during the first 10 min. This initial phase of amylase release was additive with the slower, sustained secretion which was stimulated by carbachol in a dose-dependent manner (Kd = 2.5 microM). Such a biphasic release pattern was not seen with other potent secretagogues (isoproterenol, dibutyryl cAMP) that are not thought to act via a Ca-dependent mechanism. These results suggest that parotid secretion patterns should be studied at a number of time points, which is feasible using the method reported herein.
Soor
S K
SK
Department of Anatomy, University of Ottawa, Ontario, Canada.
Hincke
M T
MT
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D002217:Carbachol / Q000494:pharmacology; D005960:Glucosides; D007202:Indicators and Reagents; D007545:Isoproterenol / Q000494:pharmacology; D007700:Kinetics; D010306:Parotid Gland / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D051381:Rats; D013056:Spectrophotometry, Ultraviolet; D000516:alpha-Amylases / Q000032:analysis / Q000378:metabolism*
D005960:Glucosides; D007202:Indicators and Reagents; D003994:Bucladesine; C036167:4-nitrophenyl maltoheptaoside; D002217:Carbachol; D000516:alpha-Amylases; D007545:Isoproterenol