TY - JOUR
T1 - Expression and characterization of a recombinant Drosophila tyramine-β-hydroxylase in silkworm infected with recombinant baculovirus
AU - Ali, Ahmed M.H.
AU - Lee, Jae Man
AU - Yoshida, Mayumi
AU - Sakashita, Kosuke
AU - Torii, Jumpei
AU - Kusakabe, Takahiro
AU - Hirashima, Akinori
N1 - Funding Information:
This study was supported, in part, by the Program for Promotion of Basic Research Activities for Innovative Biosciences (PROBRAIN) , a grant from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Integrated research project for plant, insect and animal using genome technology INSECT-1201 ) and a Grant-in-Aid (nos. 22248003 and 22248004 ) from the Japan Society for the Promotion of Science . We thank Chisa Aoki (Kyushu University Graduate School) for providing the Bme21 cell line. A. Ali was financially supported by the Global 30 Project of Kyushu University .
PY - 2012/12
Y1 - 2012/12
N2 - The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (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 d17 B. 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 OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3. H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.
AB - The insect nervous system contains biogenic amines such octopamine (OA), which is synthesized from tyramine (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 d17 B. 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 OA were 7.6, 25 °C, and 30 min, respectively. The inhibitory experiments using various concentrations of 1-(2-methoxy-5-methylphenyl) imidazole-2(3. H)-thione (MMIT) showed that MMIT inhibited DmTβH dose-dependently and that this method can be applied for screening DmTβH inhibitors.
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U2 - 10.1016/j.aspen.2012.06.004
DO - 10.1016/j.aspen.2012.06.004
M3 - Article
AN - SCOPUS:84864056563
SN - 1226-8615
VL - 15
SP - 567
EP - 572
JO - Journal of Asia-Pacific Entomology
JF - Journal of Asia-Pacific Entomology
IS - 4
ER -