Determination of peptidylglycine α-amidating monooxygenase activity in human serum by thin-layer chromatography

✍ Scribed by Noriyuki Miyazaki; Tomihiko Uemura

• Book ID: 102986478
• Publisher: Elsevier Science
• Year: 1991
• Tongue: English
• Weight: 539 KB
• Volume: 197
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(91)90364-y

✦ Synopsis

We developed a simple assay system for the quantitative evaluation of peptidylglycine alpha-amidating monooxygenase activity using as substrate a 125I-labeled synthetic tripeptide, 125I-D-Tyr-Val-Gly, thin-layer chromatography, and a radiochromatoscanner. The basic principle of this method is that thin-layer chromatography separates the reaction product, 125I-D-Tyr-Val-NH2, from the substrate in an assay mixture. The 125I activities of both substrate and product separated from each other on a thin-layer chromatography plate were quantified with a radiochromatoscanner and the rate of conversion of the substrate to the product was calculated from their counts. Human serum was used as an enzyme source and the values of alpha-amidation activity obtained by our method under optimal conditions were almost identical to those of the published method using ion-exchange chromatography (sulphopropyl-Sephadex C-50 column) and a gamma-counter. Our method makes it possible to estimate the 10-pmol level of the product using 10 microliters of human serum and to assay a large number of samples rapidly and easily. It is therefore thought to be very useful for screening various tissues for alpha-amidation activity.