Bovine γ‐Glutamylamine cyclotransferase purification

Abstract

γ-Glutamylamine cyclotransferase (γ GACT) from bovine kidney is a soluble enzyme believed to be involved in the metabolism of transglutaminase products. This enzyme catalyses the cleavage of Nε-(γ-glutamyl)lysine with intramolecular cyclization of the glutamyl moiety into 5-oxoproline and release of free lysine. Fluorescence derivatization of the lysine produced and SDS-PAGE are used to monitor the enzyme during the purification. In the initial purification of γ GACT from kidney tissue ultracentrifugation, anion exchange chromatography on DEAE-Sepharose, ammonium sulfate fractionation, and size exclusion chromatography on Sephacryl S-100 are employed. Size exclusion chromatography on Superdex-S75 using reducing conditions gives a single peak of enzyme activity with a molecular mass of ~ 20 KDa. In chromatography with strong anion exchange on a Mono Q column several peaks of activity are observed; the relative concentration of the two main peaks of activity depends on the reducing conditions. Heterogeneity of the enzyme activity is also observed in isoelectric focusing (IEF). This research is supported by a grant from the Baylor University Research Committee.