| Project/Area Number |
09680776
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Kansai Medical University |
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Principal Investigator |
KITAGAWA Kaori Kansai Medical Universtiy, research associate, 医学部, 助手 (10165813)
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| Project Period (FY) |
1997 – 1998
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| Project Status |
Completed (Fiscal Year 1998)
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| Budget Amount *help |
¥3,300,000 (Direct Cost: ¥3,300,000)
Fiscal Year 1998: ¥1,100,000 (Direct Cost: ¥1,100,000)
Fiscal Year 1997: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | Cl^- Pump / rat brain microsomal fraction / cloning / chloride ion / Northern blot / Cl^-pump / Cl^- pump |
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| Research Abstract |
Cl^- pump-rich microsomal fractions were prepared from the rat brain, solubiized using0.5% decanoyl-N-methylgluconate (MEGA-10), and applied to high performance liquid chromatography (HPLC) with an anion exchanger Mono-Q column. The C^- pump-rich fractions of HPLC eluates were electrophoresed in a 5-10% gradient polyacrylamide gel. We obtained 520 and 580 kDa protein complexes with higher activities of Cl^- pump and C^--ATPase. The sodium dodecyl sulfate-polyacrylamide gel elecWophoresis of 520 kDa protein complex yielded four protein bands at 62, 60, 55 and 51 kDa. The N-terminal sequence of 55 and 51 kDa protein determined by Edman degradation. They were unknown peptides. The primer for 55 kDa protein was designed based on a part of N-terminal sequence of 55 kDa, and the synthesis of the 5' region of cDNA was carried out from the rat brain cDNA library using polymerase chain reaction (PCR). We found a 5' region of cDNA sequence (173bp) of 55 kDa containing a possible signal peptide sequence. This sequence probe was used for the isolation of 55 kDa protein cDNA clones from the rat brain cDNA library and for Northern blot analysis. We found that the size of the mRNA of 55 kDa protein was 4.2 kb and that the mRNA was expressed mainly in the brain, and also in the liver, kidney and skeletal muscle.
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