The development of two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity.
Two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity have been developed. The assays are based on hydrolysis of the quenched-fluorimetric substrate <Glu-His-Pro-7-amino-4-methylcoumarin. Following the removal of the N-terminal <Glu by pyroglutamyl aminopeptidase type-II, liberation of 7-amino-4-methylcoumarin from the metabolite His-Pro-7-amino-4-methylcoumarin is catalyzed by one of two methods: (i) the addition of partially purified bovine serum dipeptidyl aminopeptidase type-IV or (ii) by incubating the reaction mixture for up to 2 h at 80 degrees C, thus promoting the nonenzymatic cyclization of His-Pro-7-amino-4-methylcoumarin to cyclo His-Pro and free 7-amino-4-methylcoumarin. Pyroglutamyl aminopeptidase type-II from bovine brain is used to establish appropriate assay conditions. These fluorimetric assays offer expeditious alternatives to the existing radiolabeled thyrotropin-releasing hormone assays for the determination of PAPII activity.
Gallagher
S P
SP
School of Biological Sciences, Dublin City University, Glasnevin, Ireland.
O'Leary
R M
RM
O'Connor
B
B
D000626:Aminopeptidases / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D003374:Coumarins / Q000032:analysis / Q000378:metabolism; D003500:Cyclization; D018819:Dipeptidyl Peptidase 4 / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013050:Spectrometry, Fluorescence
D003374:Coumarins; D009842:Oligopeptides; C107568:glutamyl-histidyl-prolyl-7-amino-4-methylcoumarin; D000626:Aminopeptidases; D018819:Dipeptidyl Peptidase 4; C023166:pyroglutamyl-peptidase II; C028743:7-amino-4-methylcoumarin; D011761:Pyrrolidonecarboxylic Acid