Development of a continuous, fluorometric coupled enzyme assay for thyrotropin-releasing hormone-degrading ectoenzyme.
Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4. 19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80 degrees C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave K(m) values for TRHMCA of 3.4 +/- 0.7 microM (n = 5) and 3.8 +/- 0.5 microM (n = 5), respectively. K(i) values determined by the discontinuous assay for TRH and TRH-OH were 35 +/- 4 microM (n = 3) and 311 +/- 31 microM (n = 5), respectively.
Kelly
J A
JA
Department of Biochemistry, Trinity College Dublin, Dublin, 2, Ireland.
Slator
G R
GR
Tipton
K F
KF
Williams
C H
CH
Bauer
K
K
D000626:Aminopeptidases / Q000032:analysis*; D000818:Animals; D001921:Brain / Q000201:enzymology; D005470:Fluorometry / Q000379:methods*; D007700:Kinetics; D008956:Models, Chemical; D011761:Pyrrolidonecarboxylic Acid / Q000031:analogs & derivatives; D013552:Swine
D000626:Aminopeptidases; C023166:pyroglutamyl-peptidase II; D011761:Pyrrolidonecarboxylic Acid