| Budget Amount *help |
¥6,700,000 (Direct Cost: ¥6,700,000)
Fiscal Year 1995: ¥2,700,000 (Direct Cost: ¥2,700,000)
Fiscal Year 1994: ¥4,000,000 (Direct Cost: ¥4,000,000)
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| Research Abstract |
Calcium-dependent protein kinase (CDPK)was purified 20,000-fold from soluble franction of Dunaliella, and characterized. Using an anti-Dunaliella CDPK antibody, cDANs encoding parts of CDPK were cloned from a Dunaliella cDNA library. A full length cDNA was isolated by using one of partial cDNAs as a probe, and is nucleotide sequence was determined. Deduced amino acid squence from the nucleotide sequence indicated that Dunaliella CDPK in a typical CDPK which has kinase domain at N-terminus, calmolulin-like domain at C-terminus and pseudosubstrate domain between them.
When subjected to hypo-/hypersomotic shock, 40-kDa protein kinases named HAP/LAP kinases, respectively, which have different substrate specificities and time courses of activation were activated. The activation accompanied with protein phosphorylation. Since an involvement of increase in cytosolic Ca^<2+> was anticipated for the activation, tobacco suspension culture cells transformed aequorin was subjected osmotic shock. Upon hypoosmotic shock, a transient increase in Ca^<2+> with a peak at 70 sec after the shock and subsequent activation of 50-, 75- and 80-kDa protein kinases was observed. The activation accompanied with protein phosphorylation suggesting the involvement of CDPK.
A CDPK cross-reacting with anti-Dunaliella CDPK was detected in a brackish water Characeae Lamprothamnium. When this alga injected the antibody into the cytoplasm was subjected to hypoosmotic shock, turgor regulation was suppressed even though an increase in cytosolic Ca^<2+> occured after the shock. This result suggests the involvement of CDPK in turgor regulation of this alga.
The results obtained in this study suggest that CDPK is tightly involved in osmotic signal transduction and osmoregulation in algae and higher plants.
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