Gamma-Glutamylcyclotransferase in tobacco suspension cultures - Catalytic properties and subcellular localization

Gamma-Glutamylcyclotransferase activity (EC 2.3.2.4) in ammonium sulfate precipitates (40-70% saturation) of extracts from cultured tobacco cells (Nicotiana tabacum L.cv.Samsun) was analyzed as liberation of 5-oxo-proline from L-gamma-glutamyl dipeptides. The enzyme was highly specific for the sulfur containing gamma-glutamyl dipeptides gamma glutamyl-L-methionine and gamma-glutamyl-L-cysteine. Maximum activity was obtained at pH 8.7 and 35 degrees C. As also observed with animal gamma-glutamylcyclotransferase, the tobacco enzyme exhibited a relatively low substrate affinity (gamma-glutamyl-L-methionine: K sub m 2.2 +- 0.4 mM). In contrast to animal gamma-glutamylcyclotransferase, the tobacco enzyme was not inhibited by the D-isomer of the substrate D-gamma-Glutamyl-L-methionine; it also did not use the D isomer as a substrate. Gamma-Glutamylcyclotransferase of tobacco cells was shown to be a soluble enzyme entirely localized in the cytoplasm. (IFU)