Toxocara canis: Molecular cloning, characterization, expression and comparison of the kinetics of cDNA-derived arginine kinase

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• 作者：Susiji Wickramasinghe ; Kouji Uda ; Mitsuru Nagataki ; Lalani Yatawara ; R.P.V.J. Rajapakse ; Yoshiya Watanabe ; Tomohiko Suzuki ; Takeshi Agatsuma
• 关键词：Interleukin-11 ; STAT3 ; Ischemia/reperfusion ; Cardioprotection ; Cytokine therapy
• 刊名：Experimental Parasitology
• 出版年：2007
• 出版时间：October 2007
• 年：2007
• 卷：117
• 期：2
• 页码：124-132
• 全文大小：1175 K

文摘

Arginine kinase (AK) is a member of a highly conserved family of phosphagen kinases. We determined the cDNA sequence of Toxocara canis AK, cloned it in pMAL plasmid and expressed it in Escherichia coli as a fusion protein with maltose-binding protein. The protein has a theoretical molecular mass of 45,376 Da and an estimated isoelectric point (pI) of 8.38. Alignment of the cDNA-derived amino acid sequence of T. canis AK with other phosphagen kinase sequences showed high amino acid identity with other nematode AKs, and phylogenetic analysis placed it as a distinct branch within a nematode AK cluster. Analysis of the N-terminus sequence of T. canis AK revealed the presence of a signal targeting peptide presumably targeting this protein to cytosol or endoplasmic reticulum (ER). T. canis AK showed high activity for l-arginine. The kinetic constants (K_m = 0.12 mM, K_cat = 29.18, and K_d = 0.23 mM) and V_max (43.76 μmol Pi/min/mg protein) of T. canis recombinant-AK were determined for the forward reaction. It also exhibited a synergism for substrate binding . Comparison of values in various arginine kinases indicates that T. canis AK has a high catalytic efficiency (248.19 s⁻¹ mM⁻¹). The present study contains the first description of arginine kinase in a zoonotic nematode. The determination of T. canis AK and its phosphagen biosynthetic pathway, which is completely different from those in mammalian host tissues, suggests this enzyme as a possible novel chemotherapy target for VLM syndrome in humans.