Determination of peptidylglycine alpha-amidating monooxygenase activity in human serum by thin-layer chromatography.
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.
Miyazaki
N
N
Department of Radioisotope, Psychiatric Research Institute of Tokyo, Japan.
Uemura
T
T
D000595:Amino Acid Sequence; D001205:Ascorbic Acid; D002855:Chromatography, Thin Layer / Q000379:methods*; D003300:Copper; D005069:Evaluation Studies as Topic; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007457:Iodine Radioisotopes; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000097:blood*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes; D009842:Oligopeptides / Q000737:chemistry; D013379:Substrate Specificity
D007457:Iodine Radioisotopes; D009097:Multienzyme Complexes; D009842:Oligopeptides; D003300:Copper; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D001205:Ascorbic Acid