| Budget Amount *help |
¥2,400,000 (Direct Cost: ¥2,400,000)
Fiscal Year 2002: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2001: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2000: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Research Abstract |
From the analysis of early stage in biogenesis of P-type ATPase using short term labeling, we revealed that
1) Ca-ATPase could associate with the β-subunit of (Na,K)ATPase in the early stage of its biogenesis.
2) The complex of Ca-ATPase and the β-subunit of (Na,K)ATPase are transiently, and dissociate within few hours.
3) As the β-subunit of (Na,K)ATPase, the β-subunit of (H,K)ATPase could also associate with Ca-ATPase, in the early stage of its biogenesis.
These findings suggested that the factor, which assists the topogennesis of Ca-ATPase in early stage of its biogenesis like β-subunits, exists, thus we searched such factor. The membrane and the soluble fraction prepared from Xenopus oocytes, were subjected western blotting analysis. In the soluble fraction, the 60 kDa band was detected with the anti β-subunit of (Na,K)ATPase polyclonal and the anti β-subunit of (H,K)ATPase monoclonal antibodies, but not with the anti β-subunit of (Na,K)ATPase monoclonal antibody. These two antibodies, the anti β-subunit of (Na1K)ATPase polyclonal and the anti β-subunit of (H,K)ATPase monoclonal, were prevented the Ca-ATPase synthesis in the Xenopus oocyte expression system, but another one, the anti β-subunit of (Na,K)ATPase monoclonal antibody, had no effect. These results shown the possibility that the 60 kDa protein might be involved in the Ca-ATPase synthesis. To clear this possibility, we tried to purify the 60 kDa protein. The soluble fraction prepared from Xenopus overy was fractionated by ion exchange column chromatography. The anti (H,K)ATPase β-subunit monoclonal antibody was used as a probe to detect the 60 kDa protein. The 60 kDa protein identified as a discrete band in SDS-PAGE was sequenced. Probably because of N-terminal blocking, we have not obtained the sequence of the 60kDa protein yet.
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