Formation of N epsilon-(gamma-glutamyl)-lysine isodipeptide in Chinese-hamster ovary cells.
N epsilon-(gamma-Glutamyl)-lysine isodipeptide was detected in a protein-free fraction of Chinese-hamster ovary cells and their culture fluid by using radioactive lysine as a tracer. The identity of the isodipeptide was established by its separation on ion-exchange chromatography, analysis by h.p.l.c. after derivatization, recovery of lysine after acidic hydrolysis or after cleavage by a specific enzyme, namely gamma-glutamylamine cyclotransferase. The amount of isodipeptide was raised (460 pmol/10(7) cells and 61 pmol/ml of culture fluid were observed as highest values) as the cell density increased. Effects of inhibitors of intracellular protein degradation have shown that the isodipeptide derives from cross-linking N epsilon-(gamma-glutamyl)-lysine bonds formed by tissue transglutaminase. Estimated half-life values of cross-linked proteins were about 3 h. gamma-Glutamylamine cyclotransferase, which may split the isodipeptide formed during the continuous turnover of cross-linked proteins, was also found in Chinese-hamster ovary cells. Isodipeptide may have been accumulated when either its generated amount is beyond the capacity of gamma-glutamylamine cyclotransferase or it is generated in cell compartments where this enzyme is not present.
Fesus
L
L
Department of Biochemistry, University School of Medicine, Debrecen, Hungary.
Tarcsa
E
E
D000818:Animals; D002455:Cell Division; D002460:Cell Line; D002852:Chromatography, Ion Exchange; D006224:Cricetinae; D003412:Cricetulus; D004151:Dipeptides / Q000378:metabolism*; D066298:In Vitro Techniques; D007976:Leupeptins / Q000494:pharmacology; D008744:Methylamines / Q000494:pharmacology; D009842:Oligopeptides / Q000494:pharmacology; D011506:Proteins / Q000378:metabolism*; D011503:Transglutaminases / Q000378:metabolism*; D005722:gamma-Glutamylcyclotransferase / Q000378:metabolism
D004151:Dipeptides; D007976:Leupeptins; D008744:Methylamines; D009842:Oligopeptides; D011506:Proteins; C001299:epsilon-(gamma-glutamyl)-lysine; C002101:chymostatin; C027451:methylamine; D011503:Transglutaminases; D005722:gamma-Glutamylcyclotransferase; C032854:leupeptin