Hydrazinolysis of α1-acid glycoprotein

Yosizawa, Zensaku; Sato, Tokutaro; Schmid, Karl

doi:10.1016/0304-4165(66)90134-6

Cited by 103 publications

(32 citation statements)

References 26 publications

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“…The SI stage was characterized by a very strong reactivity with HP, indicating that a large number of terminal GNAc residues appear at this point (13). This finding was corroborated by methylation analysis since 3,4,6-tri-O-methyl-GNAc was shown to be present ( Me-a-D-GNAc carbohydrate moiety of CEA was released by treatment with anhydrous hydrazine (27). After removal of excess reagents and reacetylation of the glucosamine residues, the crude preparation showed a clearly demonstrable inhibition of the CEA anti-CEA reaction in radioimmunoassay.…”

Section: Methodsmentioning

confidence: 62%

See 1 more Smart Citation

Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Hammarström

Engvall

Johansson

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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Section: Methodsmentioning

confidence: 62%

“…Hydrazinolysis of CEA was performed with anhydrous hydrazine as described by Yosizawa et al (27) (4).…”

Section: Methodsmentioning

confidence: 99%

Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Hammarström

Engvall

Johansson

et al. 1975

Proc. Natl. Acad. Sci. U.S.A.

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“…An aliquot was subjected to hydrazinolysis by heating with anhydrous hydrazine (containing catalytic amounts of hydrazine sulfate) at 105 C for 10 h as described by Yosizawa et al (30). The resulting hydrazinolysate was repeatedly evaporated with H20 to remove the hydrazine and then subjected to Bio-Gel P-4 gel filtration, as described for the lipid-linked oligosaccharides.…”

Section: Methodsmentioning

confidence: 99%

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Bailey

Deluca²,

Dürr³

et al. 1980

Plant Physiol.

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Membrane preparations from developing soybean (var. Prize) cotyledon tissue, at the time of synthesis of storage glycoproteins, catalyze the sequential assembly of lipid-linked oligosaccharides from uridine-5'-diphospho-N-acetyl-E-16_3Hlglucosamine and guanosine-5'-diphospho-n-IU-14Clmannose. The maximum size of lipid-linked oligosaccharide that accumulates contains the equivalent of 10 saccharide units on the basis of Bio-Gel P-2 gel filtration studies. These lipid-linked oligosaccharides show similar characteristics to polyisoprenyl diphosphate derivatives on diethylaminoethyl-cellulose chromatography and are potential intermediates in glycoprotein biosynthesis in this tissue. These glycolipids do not appear to turn over in pulse-chase experiments and no completed storage glycoproteins were detected among the products of these incubations. (2) and french bean (9, 12, 13) cotyledon tissue. Dolichyl phosphate has been purified from soybean and shown to act as an effective acceptor for glycosyl transfer from sugar nucleotides (5,22).Evidence that lipid-linked oligosaccharides can be assembled in a sequential manner from UDP-GlcNAc and GDP-mannose by membrane preparations from developing soybean cotyledons and that some of these, as in mammalian systems, may be involved in the synthesis of endogenous storage glycoproteins is presented here. Earlier studies in these laboratories have been concerned mainly with the assembly of glycolipids in growing tissue (pea stem) where glycoproteins do not accumulate (1, 10), and it was of particular interest to employ similar techniques with a tissue where a major part of metabolism is geared to the synthesis and deposition of glycoprotein. MATERIALS AND METHODSPlant Material. Soybean (Glycine max. var. Prize) was grown in Vermiculite with a 12-h photoperiod at 28 C (daytime) and 22 C (night). Plants were inoculated with Rhizobium japonicum and watered with N-free medium as described previously (27). Cotyledons were harvested when their fresh weight was between 100 and 250 mg.Membrane Isolation and Incubation. A membrane preparation was obtained from developing soybean cotyledons by modification of the method previously used in studies with pea stem tissue (1). The cotyledons were homogenized with a mortar and pestle at 4 C in 0.1 M Tris-HCl (pH 7.4) containing 5 mm dithioerythritol, 10 mM MgCl2 and 0.4 M sucrose, and the brei was filtered through Miracloth (Calbiochem). The filtrate was centrifuged at low speed (lOOOg for 10 min) and the pellet was dispersed for 20 s using a tissue homogenizer (Polytron, Brinkmann Instruments, at halfmaximum force). The homogenate was recentrifuged (lOOOg for 10 min) and the supernatant was combined with that from the previous centrifugation. A total membrane preparation was obtained from this supernatant by centrifugation at high speed (48,000g for 60 min).

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“…The peptide moiety of PSK was decomposed by dissolving in anhydrous hydrazine and heating at 100˚C for 2 h in accordance with identification scheme (15). Competitive ELISA was performed according to the above procedures.…”

Section: Binding Of Anti-psk Mab To Protein-degraded Pskmentioning

confidence: 99%

Anti-protein-bound polysaccharide-K monoclonal antibody binds the active structure and neutralizes direct antitumor action of the compound

Hoshi¹

2011

Oncol Rep

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Abstract. Protein-bound polysaccharide K (PSK) is extracted and purified from Coriolus versicolor (CM101), and is used as an anti-cancer agent. In this study, focusing on the direct actions of PSK, we investigated whether PSK reaches tumor and immune tissues with its active structure remaining intact, and the direct action of PSK was evaluated by its antitumor effects against MethA fibrosarcomas implanted in immunodeficient NOD/SCID mice. The results obtained suggest that PSK reaches the tumor tissue in its active form and exhibits antitumor effects against MethA cells. IntroductionProtein-bound polysaccharide-K (PSK) is a proteinpolysaccharide complex extracted from the cultured mycelia of Coriolus versicolor (strain CM101). In Japan, this compound is mainly used as an anti-cancer agent for postoperative gastric cancer and colorectal cancer in combination with chemotherapeutic agent. The molecular weight of PSK averages approximately 100,000, and varies over a wide range from 5,000 to 300,000. The main structural constituent is polysaccharide with the main chain linked by ß-D-(1➝4) bond and the side chains by ß-D-(1➝3) and ß-D-(1➝6) bonds.The antitumor effect of PSK is manifested by three major mechanisms of action: i) inhibition of cell proliferation (cytostatic effect); ii) neutralization of immunosuppressive substances; and iii) immunostimulatory activities (1-13).However, no data has hitherto demonstrated that the physiologically active structure of PSK is actually absorbed from bowel and reaches tumor and immune tissues. In addition, it remains unknown which moiety of the PSK molecule is responsible for these direct and indirect antitumor actions. One of the reasons is that PSK is not a compound with homogeneous structure, but is a mixture of glucan and protein complexes formed during the extraction process. Therefore isolation and structural determination of the active structure by conventional methods such as high performance liquid chromatography and mass spectral analysis are difficult. Furthermore, since the structure of its active site is unknown, there are difficulties in investigating the blood concentration and tumor tissue distribution of the compound after administration.Methods for the detection of PSK include the Limulus test that detects ß1-3 glucan or lipopolysaccharide (LPS) (unpublished data). However, since the Limulus test is positive not only for PSK but also for other polysaccharides containing ß1-3 glucan (such as laminarin and yeast glucan), it is not a specific detection method for PSK (unpublished data). Production of antibodies against PSK and the evaluation of these antibodies have also been reported (14). However, the rabbit anti-PSK polyclonal antibodies produced recognize all molecules containing ß1-3 glucan, ß1-4 glucan and ß1-6 glucan structures, and is therefore relatively low specificity for PSK. These antibodies are not appropriate for the detection of PSK with physiological activities (unpublished data).In the present study, we produced a monoclonal antibody against the ...

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Hydrazinolysis of α1-acid glycoprotein

Cited by 103 publications

References 26 publications

Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Nature of the tumor-associated determinant(s) of carcinoembryonic antigen.

Involvement of Lipid-linked Oligosaccharides in Synthesis of Storage Glycoproteins in Soybean Seeds

Anti-protein-bound polysaccharide-K monoclonal antibody binds the active structure and neutralizes direct antitumor action of the compound

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