| Budget Amount *help |
¥3,800,000 (Direct Cost: ¥3,800,000)
Fiscal Year 2005: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2004: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
Sarco(endo)plasmic reticulum Ca^<2+>-ATPase (SERCA) transport Ca^<2+> ions from cytoplasm into lumen coupled with ATP hydrolysis, and has essential roles in Ca^<2+> homeostasis. The Ca^<2+> transport sites are located in transmembrane domain, while catalytic site is in the three cytoplasmic domains (A,P, and N). P-domain is phosphorylated with ATP bound to N-domain. Biochemical studies on the intermediates and their analogs indicated that the large rotation of A-domain and its tight association with P-domain occur during the conformational transition of the phosphorylated intermediate (E1P to E2P) and Ca^<2+> release. 1.The author found that Tyr^<122> on the loop connecting A-domain and the 2^<nd> transmembrane helix and other hydrophobic six residues are critical for the hydrophobic interactions between A- and P-domains in the transition and hydrolysis of E2P. 2.The structural natures of stable analogs for E2P of Ca^<2+>-ATPase, i.e. E2BeF,E2A1F, and E2MgF, were explored and compared with actual E2P. It was suggested that the change in hydrophobic nature around phosphorylation site is associated with the change in phosphate geometry from BeF_<3^-> to AlF_3 or AlF_<4^-> and further to MgF_<4^<2->>. Such change likely rearranges transmembrane helices to prevent leakage of lumenal Ca^<2+>. 3.Possible functional abnormalities in three different Darier disease-causing SERCA2b mutants, I274V,L321F, and M719I. Essentially normal expression levels and only slightly reduced Ca^<2+> transport activities of I274V and M719I as compared with wild type suggest that physiological requirement for Ca^<2+> homeostasis in keratinocytes to avoid haploinsufficiency is very strict. The insensitivity to lumenal Ca^<2+> in L321F could possibly be associated with the neuropsychiatric disorder in the pedigee.
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