Shigeaki Takagi

Occurrence of free N-glycans in pea (Pisum sativum. L) seedlings

Free N-glycans have been found in pea seedlings. These free N-glycans were coupled with 2-aminopyridine and purified by gel filtration, Con Α-Sepharose affinity chromatography, and size fractionation HPLC. These structures of pyridylaminated free N-glycans were analyzed by exomannosidase digestions and ion- spray tandem mass spectrometry. The structural analyses showed that the several oligomannose-type sugar chains having one GlcNAc residue at the reducing-end side occur in the seedlings, suggesting the endo-β-N-acetylgIucosaminidase PS [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 228-232 (1996)] should be involved in the release of oligomannose-type N-glycans from the storage glycoproteins [Y. Kimura et al., Biosci. Biotech. Biochem., 60, 1841-1850 (1996)] during the germination of pea seeds.

In vitro hydrolysis of oligomannose-type sugar chains by an α-1,2-mannosidase from microsomes of developing castor bean cotyledons

An alpha-1,2-mannosidase involved in the processing of N-linked oligosaccharides was prepared from the microsomal fraction of developing castor bean cotyledons. The processing alpha-mannosidase was solubilized with 1.0% Triton X-100 and purified by ion-exchange chromatography followed by two gel filtration steps. The enzyme obtained could convert Man9GlcNAc2-PA to Man5GlcNAc2-PA, but this enzyme was inactive with Man5GlcNAc2-PA, Man4GlcNAc2-PA, and p-nitrophenyl-alpha-D-mannopyranoside. The enzyme was optimally active between pH 5.5-6.0. The processing mannosidase was inhibited by deoxymannojirimycin, EDTA, and Tris ions but not by swainsonine. Structural analyses of the mannose-trimming intermediates produced by the alpha-mannosidase revealed that specific intermediates were formed during conversion of Man9GlcNAc2-PA to Man5GlcNAc2-PA.

Interaction of Lutein with Ovalbumin and Other Proteins: Association and Acquisition of Novel Optical Activity

Lutein bound to some proteins to form complexes soluble in aqueous solutions. The formation of complexes was accompanied by novel changes in the spectroscopic properties of lutein in the visible wavelength region. The absorption peak with a maximum at 446 nm and two sub-peaks at 423 nm and 474 nm in ethanol coalesced to form a single peak at 384 nm without fine structures. This lutein with virtually no optical activity in the visible region in organic solvents showed a circular dichroic spectrum which was roughly point-symmetrical with respect to the wavelength of the maximum in absorption spectrum.These spectroscopic properties indicated that lutein molecules two or more in number form an aggregate with chirality in their mutual arrangement in space. Further addition of lutein led to the formation of red precipitates. The molar ratio of lutein-to-ovalbumin in the soluble complex obtained at a mixing ratio of 0.45 was 8, while that of the precipitate rose from 25 to 35 with a higher mixing ratio. Eighty-f...