Abstract
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 70kDa 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 70kDa. 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 30min, 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.
The inhibitory experiment showed that MMIT inhibited DmTβH dose dependently and this method can be applicable for screening of DmTβH inhibitors. [Display omitted]
► We purify 70kDa DmTβH after injection of recombinant BmNPV into silkworm. ► Western blot analysis exhibits 70kDa immunoreactive band. ► The products formed by enzyme reaction are detected by HPLC. ► The optimum pH, temperature and incubation time are 7.6, 25°C and 30min. ► This method can be applicable for screening of DmTβH inhibitors.