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

Reference ID

482

Title

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

Source

The Journal of biological chemistry, 2000, vol. 275, pp. 129-134

Authors (5)

Chang-T	Kuo-M-C
Khoo-K-H	Inoue-S
Inoue-Y

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.