Arginine kinase from Litopenaeus vannamei: cloning, expression and catalytic properties.
Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the full-length cDNA of AK was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Escherichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps.
Yao
Cui-Luan
CL
Key Laboratory of Science and Technology for Aquaculture and Food Safety of Fujian Province University, Fisheries College/Fisheries Biotechnology Institute, Jimei University, Xiamen 361021, China.
Ji
Pei-Feng
PF
Kong
Peng
P
Wang
Zhi-Yong
ZY
Xiang
Jian-Hai
JH
D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001122:Arginine Kinase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006434:Hemocytes / Q000201:enzymology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology; D033561:Penaeidae / Q000201:enzymology* / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism
D000276:Adjuvants, Immunologic; D008070:Lipopolysaccharides; D011994:Recombinant Proteins; D001122:Arginine Kinase