Arginine kinase with substrate specificity towards D-arojnine

测定了印度沙贝（Sabellastarte indica）两种精氨酸激酶（AK 1，AK 2）的cDNAs序列，并将其克隆到pMAL质粒中，在K col中表达。系统发育分析表明，印度沙贝（Sabellastarte indica）的AKs是由CK相关基因进化而来的，而不是通常的AK基因。重组Sabellastarte AK 1对各种胍类化合物表现出广泛的特异性，而Sabellastarte AK 2主要对D-和L-精氨酸表现出更强的活性，这是一种非常独特的底物特异性，以前在普通AKs中没有见过。从黄貂鱼体壁肌肉组织中分离出胍基化合物，主要成分为D-精氨酸，含量为4.85± 0.51mmol/kg。从这些结果中，我们强烈建议，在Sabellastarte，D-精氨酸是主要的磷酸化底物和AK 2对D-精氨酸的底物特异性，催化D-精氨酸的磷酸化。动物体内还存在大量其他磷酸原激酶。这些酶中的一些仅在环节动物和密切相关的组中发现，包括甘氨酰胺激酶（GE）、龙涎香碱激酶（LK）、牛磺酰胺激酶（TK）和仅限于环节动物的独特精氨酸激酶（AK）。为了更好地了解CK亚型与GK、环节动物AK、LK和TK的关系，我们确定了以下phsophagen激酶基因的内含子/外显子结构：黑刺蛾LK、多毛类Sabellastarte AK和多毛类Arenicola线粒体TK（MiTK）。对多毛类Capitella sp.的基因组数据库的分析产生了两个推定的LK基因（细胞质LK和线粒体LK（MiLK））。MiCK和环节动物磷酸酶的内含子/外显子组织的巨大相似性，加上推导的氨基酸序列的系统发育分析，支持MiCK基因是环节动物特有的磷酸酶（LK，环节动物AK，GK，MiTK和MiLK）的基础和祖先的观点。

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.