Abstract
Octopamine (OA) and tyramine (TA) are biogenic amines that act as neurotransmitters, neurohormones, and neuromodulators in the invertebrate nervous system. Tyramine-β-hydroxylase (TβH) catalyzes the biosynthesis of OA from TA. In this study, cDNA encoding Bombyx mori TβH (BmTβH) was cloned from the brain of the silkworm B. mori. The BmTβH mRNA comprised 2204 nucleotide residues and contained an open reading frame encoding 592 amino acids. The deduced amino acid sequence shared homology to several proteins belonging to the insect TβH family. Functional expression of the cloned cDNA was obtained using a B. mori baculovirus expression vector system. Western blot analysis revealed an immunoreactive band with a molecular mass of ~67.4kDa. Reverse-phase high-performance liquid chromatography (HPLC) was used to identify the products formed during incubation of the enzyme reaction mixture. The optimum pH and temperature for the conversion of TA to OA were 7.5 and 25°C, respectively. During incubation, the reaction was linear for the first 30min at 25°C and pH7.5. Inhibitory experiments carried out with various concentrations of an inhibitor showed that this method can be used for screening of BmTβH inhibitors.
The inhibitory experiments using various concentrations of an inhibitor show that BmTβH is inhibited dose-dependently. [Display omitted]
•BmTβH is purified after injection of recombinant BmNPV into silkworm cell line.•Western blot analysis exhibits ~67.4kDa immunoreactive band.•The products formed by enzyme reaction are detected by HPLC.•The optimum pH and temperature are 7.5 and 25°C; the reaction was linear for the first 30min.•This method can be applicable for screening of BmTβH inhibitors.