| Project/Area Number |
62580127
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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 |
物質生物化学
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| Research Institution | Kyushu University |
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Principal Investigator |
HIMENO Masaru Faculty of Pharmaceutical Sciences, Kyushu University, 薬学部, 助教授 (50037602)
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| Project Period (FY) |
1987 – 1988
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| Project Status |
Completed (Fiscal Year 1988)
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| Budget Amount *help |
¥1,600,000 (Direct Cost: ¥1,600,000)
Fiscal Year 1988: ¥400,000 (Direct Cost: ¥400,000)
Fiscal Year 1987: ¥1,200,000 (Direct Cost: ¥1,200,000)
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| Keywords | Lysosomal membrane / Sialoglycoprotein / クローニング / トライトゾーム / ノーザンブロット / リソゾーム膜糖蛋白質 |
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| Research Abstract |
To study the mechanism of lysosome formation, i attempt in this project to clone the cDNA encoding the sialoglycoprotein(LGP 107) which was purified from rat liver lysosomal membranes.
A rat liver cDNA library constructed with gtll as vector was screened with anti-LGP 107 antibodies, resulting in the isolation of three positive clones from approximately 2.1x10^5 phages. The longest cDNA among the three clones was adopted for its sequencing. The sequence thus determined, Ala-Pro-Ala-Leu-Phe-Glu-Val-Lys-Asp-Asn, was found to be identical with the NH_2-terminal sequence deduced from the nucleotide sequence. It was thus evident that the longest cDNA codes for the entire length of mature LGP 107 but lacks the segment encoding a NH_2-terminal, cleavable signal peptide. The termination codon TAG (nucleotides 1161-1163) was followed by the 694-nucleotide 3'-noncoding region and poly(A) tail. As is the case for lysosomal membrane glycoproteins from other sources, the primary structure of rat LGP 107 contains a potential hinge region (residues 169-191) that is rich in proline and serine and thought to separate the polypeptide into two domains. The primary structure of LGP 107 also contains 20 potential asparagine-linked glycosylation sites and 8 cysteine residues at the same spacings. It is clear that the much higher molecular mass estimated for purified LGP 107 by SDS-PGE (107 KDa) than that predicted here (41.9 KDa) is due to the presence of sugar chains in the former. LGP 107 possessses a 24-residue strongly hydrophobic stretch (residues 351-375) near the COOH-terminus. It is highly likely that this stretch serves as a transmembrane segment and, if so it is expected that the remaining short COOH-terminal portion (residue 375 to the COOH-terminus) is extruded to the cytoplasm.
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