Developmentally regulated expression of a peptide:N-glycanase during germination of rice seeds (Oryza sativa) and its purification and characterization.

Abstract

Peptide:N-glycanase (PNGase; EC 3.5.1.52) activity was detected in dormant rice seeds (Oryza sativa) and the imbibed rice grains. Time-course studies revealed that the enzyme activity remained almost constant until about 30 h after imbibition in both of endosperm- and embryo tissue-containing areas, and started to increase only in growing germ part, reached a peak at about 3-day stage, followed by a gradual decrease concomitant with a sharp increase in the coleoptile. The specific activity increased about 6-fold at about 3-day stage. PNGase was purified to electrophoretic homogeneity from the extracts of germinated rice seeds at 24 h, and the apparent molecular weight of the purified enzyme, estimated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), was about 80,000. The purified enzyme was designated PNGase Os to denote its origin. The N-terminal sequence of the 10 residues was determined to be SYNVASVAGL. The purified PNGase Os in SDS-PAGE appeared as a rather broad band, consistent with the presence of multiple glycoforms as indicated by chromatographic behavior on a Sephadex G-75 column. PNGase expressed in coleoptile under anoxia condition was also purified, and both of the purified enzymes were found to exhibit very similar, if not identical, electrophoretic mobility in SDS-PAGE. PNGase Os exhibited a broad pH-activity profile with an optimum of 4-5 and, interestingly, was significantly inactivated by K(+) and Na(+) at near the physiological concentration, 100 mM. These results are discussed in relation to other work.

Statistics

0100200'03'05'07'09'11'13'15'17
Citations per Year

266 Citations

Semantic Scholar estimates that this publication has 266 citations based on the available data.

See our FAQ for additional information.

Cite this paper

@article{Chang2000DevelopmentallyRE, title={Developmentally regulated expression of a peptide:N-glycanase during germination of rice seeds (Oryza sativa) and its purification and characterization.}, author={T E Chang and M. C. Kuo and Kian Hoe Khoo and Satoshi Inoue and Yusuke Inoue}, journal={The Journal of biological chemistry}, year={2000}, volume={275 1}, pages={129-34} }