| Budget Amount *help |
¥3,850,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥150,000)
Fiscal Year 2007: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000)
Fiscal Year 2006: ¥800,000 (Direct Cost: ¥800,000)
Fiscal Year 2005: ¥2,400,000 (Direct Cost: ¥2,400,000)
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| Research Abstract |
We determined the cDNA-derived amino acid sequences of two arginine kinases (AK1, AK2) from the annelid Sabellastarte indica, cloned the cDNAs into pMAL plasmid and expressed them in K coll. The phylogenetic analyses suggested that Sabellastarte AKs have evolved from a CK-related gene, not from the usual AK gene. The recombinant Sabellastarte AK1 showed a broad specificity towards various guanidine compounds, while the Sabellastarte AK2 mainly showed stronger activity for both D- and L-arginine, a very unique substrate specificity not seen before in usual AKs. We isolated guanidino compounds from the body wall musculature of Sabellastarte, and found that the major compound is D-arginine with a concentration of 4.85±0.51 mmol/kg. From these results, we suggest strongly that in Sabellastarte, D-arginine is the major phosphagen substrate and that the AK2 with substrate specificity towards D-arginine, catalyzes the phosphorylation of D-arginine. A broad array of other phosphagen kinases are present in animals. Some of these enzymes are found only in annelids and closely related groups including glyocyamine kinase (GE), lombricine kinase (LK), taurocyamine kinase (TK) and a unique arginine kinase (AK) restricted to annelids. To gain a greater understanding of the relationship of the CK isoforms to GK, annelid AK, LK and TK, we have determined the intron/exon organization of the genes for the following phsophagen kinases : earthworm Eisenia LK, polychaete Sabellastarte AK, and polychaete Arenicola mitochondrial TK (MiTK). Analysis of genomic database for the polychaete Capitella sp. yielded two putative LK genes (cytoplasmic LK and mitochondrial LK (MiLK)). The great similarity in intron/exon organization of MiCKs and the annelid phosphagen kinases, coupled with phylogenetic analysis of deduced amino acid sequences, support the view that the MiCK gene is basal and ancestral to the phosphagen kinases (LK, annelid AK, GK, MiTK and MiLK) unique to annelids.
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