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Ahmed M.H. Ali,Nazmul Hasan,Renkai Guo,Hiroto Ohta,Akinori Hirashima 한국응용곤충학회 2014 Journal of Asia-Pacific Entomology Vol.17 No.3
Octopamine (OA) and tyramine (TA) are biogenic amines that act as neurotransmitters, neurohormones, andneuromodulators in the invertebrate nervous system. Tyramine-β-hydroxylase (TβH) catalyzes the biosynthesisof OA from TA. In this study, cDNA encoding Bombyx mori TβH (BmTβH) was cloned from the brain of the silkwormB. mori. The BmTβH mRNA comprised 2204 nucleotide residues and contained an open reading frameencoding 592 amino acids. The deduced amino acid sequence shared homology to several proteins belongingto the insect TβH family. Functional expression of the cloned cDNA was obtained using a B. mori baculovirus expressionvector system. Western blot analysis revealed an immunoreactive band with a molecular mass of~67.4 kDa. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the productsformed during incubation of the enzyme reaction mixture. The optimumpH and temperature for the conversionof TA to OA were 7.5 and 25 °C, respectively. During incubation, the reaction was linear for the first 30 min at25 °C and pH 7.5. Inhibitory experiments carried out with various concentrations of an inhibitor showed thatthis method can be used for screening of BmTβH inhibitors.
Ahmed M.H. Ali,Jae Man Lee,Mayumi Yoshida,Kosuke Sakashita,Jumpei Torii,Takahiro Kusakabe,Akinori Hirashima 한국응용곤충학회 2012 Journal of Asia-Pacific Entomology Vol.15 No.4
The insect nervous systemcontains biogenic amines such octopamine (OA),which is synthesized fromtyramine (TA) by catalysis of tyramine-β-hydroxylase (TβH). In this study, the Drosophila 70 kDa tyramine-β-hydroxylase (DmTβH) protein was purified after the recombinant nucleopolyhedrovirus isolated from Bombyx mori (BmNPV) containing the TβH gene was injected into the hemocoel of the fifth instar larvae from the d17B. mori strain. Western blot analysis revealed an immunoreactive band with a molecular mass of 70 kDa. The products formed by incubating the enzyme reaction mixture were separated and detected by reverse phase high-performance liquid chromatography. The optimum pH, temperature, and incubation time for the conversion of TA to OAwere 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.