Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China.
Dev Comp Immunol. 2012 Jun;37(2):270-8. doi: 10.1016/j.dci.2012.03.008. Epub 2012 Apr 3.
Arginine kinase (AK) catalyzes the reversible phosphorylation of l-arginine to form phosphoarginine, and plays a critical role in energy metabolism in invertebrates. In the present study, a scallop AK gene was identified from Chlamys farreri with an open reading frame (ORF) of 1101bp encoding for a protein of 366 amino acids (designed as CfAK). An ATP-gua PtransN domain which was described as a guanidine substrate specificity domain (GS domain) and an ATP-gua Ptrans domian which was responsible for binding ATP, were both identified in CfAK. The mRNA transcripts of CfAK were detectable in haemocytes, hepatopancreas, adductor muscle, mantle, gill, kidney and gonad, with the highest expression level in the muscle and the lowest level in the hemocytes. The expression level of CfAK mRNA increased from fertilized eggs to eyebot, and reached the highest in the trochophore stage. The relative expression level of CfAK mRNA in muscle was up-regulated significantly after LPS (0.5mg/mL) stimulation, and reached the peak at 6h (5.2-fold, P<0.05). The activity of inducible nitric oxide synthase (iNOS) in the supernatant of muscle homogenate increased significantly from 3.2U/mg at 0 h to 9.7 U/mg at 12h after LPS stimulation, while the concentration of nitric oxide (NO) in the supernatant of muscle homogenate began to increase at 3h (21.55 μmol/L), and reached the top concentration at 24h (42.27 μmol/L), then recovered to the normal level after 48 h. The recombinant protein of CfAK (rCfAK) expressed in Escherichia coli displayed Arginine kinase activity, and its apparent K(m) was 0.82 ± 0.11 and 1.24 ± 0.13 mM for L-arginine and ATP-Na, respectively. The results indicated that the CfAK was involved in energy production and utilization during the whole life process, and might refer to the immunomodulation process via altering the NO concentration and iNOS activity in scallop Chlamys farreri.
精氨酸激酶（AK）催化 l-精氨酸的可逆磷酸化，形成磷酸精氨酸，在无脊椎动物的能量代谢中发挥关键作用。本研究从栉孔扇贝中鉴定出一种 AK 基因，其开放阅读框（ORF）为 1101bp，编码 366 个氨基酸的蛋白质（命名为 CfAK）。CfAK 中鉴定出一个 ATP-胍 P 转移 N 结构域，该结构域被描述为胍底物特异性结构域（GS 结构域）和一个负责结合 ATP 的 ATP-胍 P 转移结构域。CfAK 的 mRNA 转录本可在血细胞、肝胰腺、闭壳肌、套膜、鳃、肾和性腺中检测到，在肌肉中的表达水平最高，在血细胞中的表达水平最低。CfAK mRNA 的表达水平从受精卵到眼点幼虫增加，在担轮幼虫期达到最高。肌肉中 CfAK mRNA 的相对表达水平在 LPS（0.5mg/mL）刺激后显著上调，在 6h 时达到峰值（5.2 倍，P<0.05）。LPS 刺激后肌肉匀浆上清液中诱导型一氧化氮合酶（iNOS）的活性从 0h 的 3.2U/mg 显著增加到 12h 的 9.7U/mg，而肌肉匀浆上清液中一氧化氮（NO）的浓度在 3h 时开始增加（21.55 μmol/L），在 24h 时达到最高浓度（42.27 μmol/L），然后在 48h 后恢复正常水平。在大肠杆菌中表达的 CfAK 重组蛋白（rCfAK）显示出精氨酸激酶活性，其对 L-精氨酸和 ATP-Na 的表观 K(m)分别为 0.82±0.11 和 1.24±0.13mM。结果表明，CfAK 参与了栉孔扇贝整个生命过程中的能量产生和利用，可能通过改变 NO 浓度和 iNOS 活性来参与免疫调节过程。
