1993 Fiscal Year Final Research Report Summary
The role of beta-COP like membrane protein in lysosome formation
| Project/Area Number |
04808031
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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 |
代謝生物化学
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| Research Institution | Juntendo University School of Medicine |
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Principal Investigator |
EZAKI Junji Juntendo University, School of Med., Instructor, 医学部, 助手 (60232948)
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| Co-Investigator(Kenkyū-buntansha) |
ISHIDOH Kazumi Juntendo Univ., School of Med., Instructor, 医学部, 助手 (40212906)
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| Project Period (FY) |
1992 – 1993
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| Keywords | beta-COP / lysosome / adaptin / PCR / lgp120 / coated vesicle |
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| Research Abstract |
Intracellular traffic between the membrane compartments of eukaryotic cells relies on the movement of vesicle carriers. Recently, it has been suggested that the coat proteins highly concentrated in non-clathrin coated vesicle share homology with the adaptin proteins of clathrin coated vesicles. Whereas the transport systems supporting the functions of lysosome are different from these traffic systems, there should be similar protein mediating vesicle carrier. We search the protein by the following methods.
1. In the pairwise comparisons, significant homology of beta-COP was detected to beta-adaptin. The pairs sequences fit for primers were selected in accordance with the conserved sequence of these protein and made degenerated primers, a mixture of oligonucleotides varying in nucleotide sequences byt have the same number of nucleotides. We are planning to do PCR amplification using these primers with loosely regulated conditions.
2. For purification of coasted vesicle adaptors, synthetic peptides that correspond to the cytoplasmic domain of lap120 (lysosomal glycoprotein of 12-kDa) were immobilized on Sepharose 4B.Proteins of 50kDa and 90kDa were partially purified from rat liver cytosolic fractions by the lgp120-Sepharose affinity chromatography. These proteins were further purified with Sephadex G200 gel filtration and DE52 ion exchange column chromatography. The partial sequence of the protein of 50kDa was determined and polyclonal antibody was made against to this protain. Studies about the role of these proteins on lysosomal formation are underway.
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