Developmentally regulated expression of a peptide:N-glycanase during germination of rice seeds (Oryza sativa) and its purification and characterization.
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.
Chang
T
T
Institute of Biological Chemistry, Academia Sinica, Taipei 115, Taiwan.
Kuo
M C
MC
Khoo
K H
KH
Inoue
S
S
Inoue
Y
Y
D000581:Amidohydrolases / Q000096:biosynthesis* / Q000302:isolation & purification; D018548:Cotyledon / Q000201:enzymology; D018506:Gene Expression Regulation, Plant; D018525:Germination / Q000502:physiology*; D009844:Oligosaccharides / Q000302:isolation & purification; D012275:Oryza / Q000201:enzymology*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D012639:Seeds / Q000201:enzymology; D016339:Spectrometry, Mass, Fast Atom Bombardment; D014018:Tissue Distribution
D009844:Oligosaccharides; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase