RESUMO
Arylalkylamine N-acetyltransferase (AANAT; EC 2.3.1.87) catalyzes the N-acetylation of arylalkylamines. A cDNA encoding AANAT (ApAANAT) was cloned from Antheraea pernyi by PCR. The cDNA of 1966 bp encodes a 261 amino acid protein. The amino acid sequence was found to have a high homology with Bombyx mori AANAT (BmNAT) but had very low homology with vertebrate AANATs. Amino acid sequence analysis revealed that four insect AANATs cloned from three species including ApAANAT formed a distinct cluster from the vertebrate group. A recombinant ApAANAT protein was expressed in Sf9 cells using a baculovirus expression system, having AANAT activity. The transformed cell extract acetylated tryptamine, serotonin, dopamine, tyramine, octopamine and norepinephrine. The AANAT activity was inhibited at over 0.03 mM tryptamine. Although insect AANATs have been considered as a target of insecticide, this type of insecticide has never been developed. Screening a chemical library of Otsuka Chemical Co., Ltd., we found a novel compound and its derivatives that inhibited the AANAT activity of ApAANAT. This may facilitate investigation of the monoamine metabolic pathway in insects and the development of new types of insecticides and inhibitors of AANATs.
Assuntos
Arilalquilamina N-Acetiltransferase/metabolismo , Desenho de Fármacos , Proteínas de Insetos/metabolismo , Mariposas/enzimologia , Acetilação , Sequência de Aminoácidos , Animais , Arilalquilamina N-Acetiltransferase/classificação , Arilalquilamina N-Acetiltransferase/genética , Baculoviridae/genética , Sequência de Bases , Biocatálise/efeitos dos fármacos , Monoaminas Biogênicas/metabolismo , Western Blotting , DNA Complementar/genética , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Vetores Genéticos/genética , Proteínas de Insetos/antagonistas & inibidores , Proteínas de Insetos/genética , Cinética , Dados de Sequência Molecular , Mariposas/genética , Praguicidas/química , Praguicidas/farmacologia , Filogenia , Homologia de Sequência de Aminoácidos , Células Sf9 , Especificidade por Substrato , Triptaminas/metabolismo , Triptaminas/farmacologiaRESUMO
Arylalkylamine N-acetyltransferase (AANAT) catalyzes N-acetylation of arylarkylamines. A cDNA of Bombyx mori insect AANAT (Bm-iAANAT) was found by searching an expressed-sequence tag (EST) database of B. mori (SilkBase). The cDNA encoded a 261 amino acid protein. The mRNA of Bm-iAANAT was expressed in eggs, larvae, adults and various tissues. Recombinant Bm-iAANAT protein was expressed in Sf9 cells by a baculovirus expression system. The AANAT activity of Bm-iAANAT was inhibited by high concentrations (over 0.01 mM) of tryptamine used as a substrate. The Bm-iAANAT acetylated tryptamine, serotonin, dopamine, octopamine, tyramine and norepinephrine. This is the first report of a cloned AANAT that acetylated norepinephrine. These results suggest that Bm-iAANAT is a novel member of insect AANAT family with unique kinetic properties and a broad substrate range.
Assuntos
Arilalquilamina N-Acetiltransferase/química , Arilalquilamina N-Acetiltransferase/genética , Bombyx/enzimologia , Bombyx/genética , Proteínas de Insetos/química , Proteínas de Insetos/genética , Animais , Arilalquilamina N-Acetiltransferase/biossíntese , Baculoviridae , Sequência de Bases , Clonagem Molecular , DNA Complementar/genética , Expressão Gênica , Proteínas de Insetos/biossíntese , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Especificidade por SubstratoRESUMO
Imaginal disc growth factor (IDGF) is a soluble polypeptide growth factor that was first identified from the conditioned medium of Drosophilia imaginal disc C1.8+ cells. Working with insulin, IDGF stimulated the growth of cultured imaginal disk cells, which suggested that IDGF might function as a cofactor of Drosophila insulin or insulin like peptide. Here we report a new member of the IDGF family, named MbIDGF, from the cabbage armyworm, Mamestra brassicae. Using a cloned cDNA of MbIDGF, recombinant MbIDGF protein was expressed in baculovirus-infected Sf9 cells and purified. Without insulin, the recombinant MbIDGF protein stimulated cell growth of SES-MaBr-4 and NIAS-MaBr-93 cell lines that were derived from the fat bodies and hemocytes of M. brassicae, in a dose-dependent manner. The saturation of growth stimulation by MbIDGF was attained for the two types of cells at 80 ng/ml (0.8 nM) and 300 ng/ml (6 nM), respectively. The results suggest that MbIDGF may stimulate the growth of lepidopteran cells by a new mechanism without associating with the insulin pathway.
