1993 Fiscal Year Final Research Report Summary
Molecular mechanism of laminin variants expression
| Project/Area Number |
03454063
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
応用生物化学・栄養化学
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| Research Institution | Nagoya University |
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Principal Investigator |
KITAGAWA Yasuo Nagoya University, Bioscience Center*, Professor, 生物分子応答研究センター, 教授 (50101168)
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| Project Period (FY) |
1991 – 1993
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| Keywords | Basement membrane / Laminin / Kalinin / Endothelial cells / Chaperones |
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| Research Abstract |
Sequence information showed that interchain hydrophobic- and ionic interactions among heptad repeats in domain I and II of the A (merosin, kalinin A or A'), B1 (s-laminin or kalinin B1) and B2 (kalinin B2) chains are the basic mechanism for the formation of various laminin isoforms. However, our in vivo analysis of the assembly, replacement and intracellular sorting of laminin subunits suggested that additional cellular factors are involved in these process.
Bovine aortic endothelial cells (BAEC ; producing AB1B2 and A'B1B2), embryonal carcinoma F9 cells (F9 ; producing AB1B2) or human keratinocytes were labelled with [^<35>S]methionine, incubated with a cross-linking reagent : dithio-bis-succinimidylpropionate (DSP) and cell lysate was immunoprecipitated and analyzed by SDS electrophoresis under reducing and nonreducing conditions.
Nonreducing electrophoresis showed marked reduction of monomeric forms of A, A', B1 and B2 and B1B2 dimer, suggesting that the unassembled froms were cross-linked with cellular proteins. Reduced electrophoresis of immunoprecipitate from BAEC showed three major bands of 80, 60 and 50 kDa in addition to laminin subunits. Same experiment with F9 cells and keratinocytes showed a band of 100 kDa and bands of 90, 70 and 50 kDa, respectively. Two dimensional electrophoresis showed many spots of monomeric A, B1, B2 and B1B2 dimer mirgating differently in nonreducing electrophoresis. This result was consistent with the hypothesis that many molecules of chaperones are associated with each monomeric or dimeric form of laminin subunits.
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