Purification of gamma-glutamylamine cyclotransferase from bovine kidney: Separation from gamma-glutamyl amino acid cyclotransferase

gamma-Glutamylamine cyclotransferase (gamma-GACT) is an intracellular enzyme found in the cytosol of cells. It catalyzes the release of free amines and 5-oxo-proline (pyroglutamic acid, 5-pyrrolidone 2-carboxylic acid) as a result of the intramolecular cyclization of L-gamma-glutamylamines. Transglutaminases catalyze the cross linking of proteins through the formation of epsilon-(L-gamma-glutamyl)-L-lysine and it has been suggested recently that these cross-links might be implicated in the mechanism of neurofibrillary tangle formation. The enzyme (gamma-GACT) has been found in several organs and tissues and epsilon-(L-gamma-glutamyl)-L-lysine is a substrate for it. The highest activity of the enzyme has been found in the kidney. A similar enzyme, gamma-glutamyl amino acid cyclotransferase (gamma-GAACT), catalyzes the intramolecular cyclization of alpha-(L-gamma-glutamyl)-L-amino acids with the release of 5-oxo-L-proline and a free L-amino acid in a reaction like that of gamma-GACT. The enzyme is similar in characteristics to gamma-GACT and both enzymes are difficult to separate when purification using conventional techniques has been attempted. The separation of the two enzymes (gamma-GACT and gamma-GAACT) from bovine kidney homogenates has been achieved using Fast Protein Liquid Chromatography (FPLC) with a Mono QRTM HR 5/5 column. The resulting gamma-glutamylamine cyclotransferase that has been purified, is free of gamma-glutamyl amino acid cyclotransferase as shown when assays using the substrates for both enzymes were conducted on the purified preparation. The current study's primary focus was to obtain more enzyme to acquire more data regarding the properties and structure of gamma-GACT. The purified enzyme is being used to analyze the cross linking of proteins since it degrades the isolated epsilon-(L-gamma-glutamyl)-L-lysine crosslink.