| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2005: ¥1,300,000 (Direct Cost: ¥1,300,000)
Fiscal Year 2004: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
To optimize the condition for expression of SERCA1a protein using COS-1 cells, three transfection methods (Aveovirus, liposome mediated, and electroporation) were tested. The amount of SERCA1a protein in the microsome prepared from transfected COS-1 cells were more than 2 % of total membrane protein in all transfection methods tested. Finally, I chose lipofection method for transfection, since it was convenient and reproducible.
It was very difficult to measure the amount of bound Ca^<2+> to expressed SERCA1a protein, since the background level was extremely and the bound Ca^<2+> was easily removed by washing. In this study, I found the method to measure the amount of bound Ca^<2+> directly by exploring washing condition.In the wild type SERCA1a, the amount of ADP-insensitive EP (E2P) in the steady state was not altered by Ca^<2+>. On the other hand the amount of E2P in the steady state of Y122A mutant SERCa1a was changed according to the Ca^<2+> concentrations in the medium. Higher Ca^<2+> concentration in the medium brought lower amount of E2P, and higher medium pH shifts Ca^<2+> dependency toward lower Ca^<2+> concentration. The apparent Ca^<2+> affinity estimated from Ca^<2+> dependency of E2P fraction were approximately 150 μM at pH 7.5.
The amounts of bound Ca in the steady state measured by the method described above were compared with the amount of ADP-sensitive EP (E1P). In the wild type, the amounts of E1P and bound Ca were well matched each other. This result shown that no phosphoenzyme except E1PCa_2 exist in the steady state of wild type SERCA1a. On the other hand, the amount of bound Ca in the steady state of Y122A mutant was significantly larger than E1P fraction. This result means that Ca-bound ADP-insensitive EP (E2PCa_x) fraction was exactly existing in the steady state of Y122A mutant.
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