A rapid fluorometric assay for N-terminal glutaminyl cyclase activity using high-performance liquid chromatography.
A rapid, sensitive method for the quantification of glutaminyl cyclase activity has been developed. The assay involves enzymatic conversion of the model peptide Gln-Leu-Tyr-Glu-Asn-Lys-epsilon-(Dns)-OH to less than Glu-Leu-Tyr-Glu-Asn-Lys-epsilon-(Dns)-OH. Both the product and substrate of this reaction are detected in a single assay in quantities as low as 100 fmol using isocratic reverse phase high-performance liquid chromatography with fluorometric detection. The method is highly reproducible and ideally suited for the rapid analysis of large numbers of samples. The applications of the assay to both the detection of glutaminyl cyclase activity during enzyme purification and the more rigorous enzymology studies dependent on the precise measurement of initial reaction velocities are demonstrated.
Consalvo
A P
AP
Department of Protein Chemistry, Unigene Laboratories, Inc., Fairfield, New Jersey 07006.
Young
S D
SD
Jones
B N
BN
Tamburini
P P
PP
D000217:Acyltransferases / Q000032:analysis*; D019881:Aminoacyltransferases; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003619:Dansyl Compounds / Q000302:isolation & purification; D066298:In Vitro Techniques; D009842:Oligopeptides / Q000302:isolation & purification; D010902:Pituitary Gland / Q000378:metabolism; D013050:Spectrometry, Fluorescence
D003619:Dansyl Compounds; D009842:Oligopeptides; D000217:Acyltransferases; D019881:Aminoacyltransferases; C059280:glutaminyl-peptide cyclotransferase