1994 Fiscal Year Final Research Report Summary
Resolution of saccharide structure and biochemical and immunological characterization of unknown glycoprotein which reacts with the antibody of milk fat globule membrane protein
| Project/Area Number |
05660129
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
食品科学・栄養科学
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| Research Institution | Utsunomiya University |
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Principal Investigator |
KANNO Choemon Utsunomiya University Applied Biochemistry Professor, 農学部, 教授 (30011969)
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| Project Period (FY) |
1993 – 1994
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| Keywords | Bovine milk / Milk fat globule membrane / Glycoprotein / Monoclonal antibody / GP-88 glycoprotein / Lectin affinity / Saccharide chain structure / Antibody affinity chromatography |
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| Research Abstract |
1. Monoclonal antibody to PAS-4 glycoprotein which is a major constituent protein of bovine milk fat globule membrane was prepared and designated KAS-4. In addition, some proteins which react with KAS-4 was found in milk whey protein and also named as GP-88. GP-88 was glycoprotein and molecular weight was estimated to 88,000 on SDS-PAGE.
2. GP-88 was purified from whey protein by affinity chromatography on Protein G and then KAS-4.Purified GP-88 was a single band on SDS-PAGE.Molecular weight of GP-88 treated with N-glycanase was estimated to 57,000. Biochemical properties of GP-88 and PAS-4 were compared.
3. In addition, epitope of KAS-4 to GP-88 and PAS-4 was examined by immunoblotting after treatment with trifluoromethanesulfonic acid and N-glycanase. Epitope was found in protein moiety.Furthermore, fragments after digestion with trypsin were analyzed on HPLC of C8 and C18 reversed columus and an active peptide to KAS-4 from both GP-88 and PAS-4 was detected by ELISA.
4. The contents of GP-88 in whey and PAS-4 in milk fat globlule membrane in laction periods were determined. It was found that GP-88 content increased the proceeding of laction, whereas PAS-4 content decreased.
5. N-Linked saccharide chains were removed from GP-88 by hydrazine hydrolysis, and the structure of sugar chains was analyzed by size estimation on HPLC,sequential hydrolysis with exoglycosidases, acetolysis, and methylation analysis on GC-MS.A representative structure was proposed as shown in the following.
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