|Budget Amount *help
¥2,000,000 (Direct Cost : ¥2,000,000)
Fiscal Year 1996 : ¥600,000 (Direct Cost : ¥600,000)
Fiscal Year 1995 : ¥1,400,000 (Direct Cost : ¥1,400,000)
Plant-origin endo-beta-N acetylglucosaminidases have been purified or partially purified from pea seeds, soybean seeds, ginkgo seeds, and immature tomato fruit. The molecular weights of endo-GB (Ginkgo biloba), endo-GM (soybean), and endo-LE (tomato) were determined to be about 64 kDa by gel-filtration or SDS-PAGE.On the other hand, the molecular weight of endo-PS (pea) was determined to be 42 kDa by MALDI TOF-MS.All theseendoglycosidases were found to have optimum pH between 6 and 7 and optimum temperature at about 35ﾟC.And these plant-origin endoglycosidases could hydrolyze the beta-1-4linkage of chitobiose in oligomannose type N-glycans but not the linkage of xylose/fucose containing complex type N-glycans. Using various oligomannose type sugar chains, the detail substrate specificity was further investigated. High mannose type structures bearing alpha1-2 mannosyl residue (s), Man9-6GlcNAc2, could be hydrolyzed at 80-100% relative hydrolysis rate. Man5-3GlcNAc2 stuructures bearing n
o alpha1-2 mannosyl residue could be also hydrolyzed by these endoglycosidase, however, the relative reaction rates were only 20-60% comparing that for Man6GlcNAc2 structure. These results suggested that (1) plant-origin endoglycosidase has a common subsite for binding or recogmizing alpha1-2 mannosyl residue (s) and the subsite may regulate the hydrolysis rate ; (2) plant-origin endoglycosidase may serve to release high mannose type N-glycans bearing alpha1-2 mannosyl residue (s)
A peptide : N-glycanase (PNGase-GM) has been purified from soybean seed to homogeneity. The molecular weight of PNGase-GM was 90 kDa by gel-filtration and SDS-PAGE.Optimum pH of PNGase-GM was arround 5.0. PNGase-GM could hydrolyze beta-aspartlglycosylamine linkage of glycopeptides having oligomannose type, hybrid type, xylose/fucose containing type, and sialic acid containing complex type sugar chains. However, the reaction rates was depend on N-glycan structures. Assuming that the relative hydrolysis rate for the glycopeptides bearing oligomannose type is 100%, the rate for glycopeptide bearing xylose/fucose containing type was 30%, and that for glycopeptides bearing sialic acid containing complex type were less than 5%.
In epicotyls and hypocotyls of pea seedlings, free N-glycans bearing high mannose type and xylose/fucose containing type structures have been identified by ion-spray tandem mass spectrometry (IS-MS/MS). This result clearly indicates that free N-glycans produced by two kinds of N-glycan releasing enzymes do occur in the developing tissues of plant cells. Less