Evidence of two enzymes performing the de-N-glycosylation of proteins in barley: expression during germination, localization within the grain and set-up during grain formation

The occurrence of two enzymes performing de-N-glycosylation of glycoprotein s, namely, endo-N-acetyl-beta-glucosaminidase (ENGase, EC 3.2.1.96) and pep tide-N-4-(N-acetyl-beta-D-glucosaminyl) asparagine amidase (PNGase, EC 3.5. 1.52) was investigated in barley, cv. Plaisant (a winter six rowed variety) . The dry grain showed both activities according to the HPLC detection of t he hydrolysis of fluorescent resorufin-labelled substrates, However, PNGase activity was 16-fold higher than ENGase activity. During germination, both activities increased, PNGase by only 1.5-fold compared to nearly 4.8-fold for ENGase over the 4 d following imbibition, The localization of these act ivities within the grain showed that the major contribution of PNGase was d ue to the endosperm, typically representing over 90% of the whole grain act ivity, In contrast, ENGase activity was especially high in the embryo and, later, in the developing plantlet (10-fold higher than in the endosperm), p articularly in the rootlets and scutellum, In developing spikes, PNGase act ivity was 5.6-fold higher than in the leaves, but similar ENGase activity w as measured in both organs. During grain formation, PNGase activity followe d dry matter increase together with endosperm development. In contrast, ENG ase activity dropped by 66% at the beginning of grain filling before stabil izing until harvest, The occurrence of de-N-glycosylation-performing enzyme s throughout the development of barley raises the question of the nature of their natural substrates. Moreover, the prevalence of one of these enzymes over the other depending on the organ and the developmental stage, could r epresent the first evidence of specific functions for each enzyme.