| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2001: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2000: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Research Abstract |
1. The functional role of Cys876 and Cys888 in the luminal loop between 7^<th> and 8^<th> transmembrane helices of sarcoplasmic reticulum calcium pump was investigated. Isolation and sequencing of disufide-containing peptides from the pepsin-digest of the calcium pump showed that a disulfide bond is formed between these cysteines. All of C876A, C888A, and C876A/C888A mutants lacked Ca^<2+> transport activity, but their ATP hydrolysis was not inhibited. These results suggest that this disulfide bond play a role in stabilizing the enzyme structure important for coupling between ATP hydrolysis and Ca^<2+> transport.
2. The cytoplasmic domains of calcium pump are small cytosolic (A), nucleotide binding (N), and phosphorylation (P) domains, which are conserved among P-type cation transporting ATPases. Movement of these domains during the ATPase reaction cycle was investigated by analyzing digestion-susceptibility of the loop connecting A with transmembrane domain (A-M loop) to proteinase K or V8 and digestion-susceptibility of A to trypsin. The results suggest that P, N, and A-M loop (but not A) gather in substrate bound enzyme (CaE/ATP) and Ca^<2+>-bound phosphoenzyme, and that all 3 domains gather to form the most compact structure by the phosphoenzyme isomerization to Ca^<2+>-unbound form (Ca^<2+>-translocating step), in which A rotate by about 90 ℃.
3. It is known that Ca^<2+>-unbound calcium pump is rapidly denatured if solubilized with detergent, so that structural study of this state has not been easy. I previously reported that Mg^<2+> and fluoride bind to the enzyme very tightly to form an analog of Ca^<2+>-unbound phosphoenzyme. It was found that purified and C12E8-solubilized Mg^<2+>/F-bound calcium pump is completely active for at least 20 days without Ca^<2+>, and that this analog may be useful for crystallization.
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