Autor(es): Jirajaroenrat, K; Pongjaroenkit, S; Krittanai, C; Prapanthadara, LA; Ketterman, AJ

Resumo: Three cDNA Sequences of glutathione S-transferase (GST), adgst1-2, adgst1-3 and adgst1-4, which are alternatively spliced products of the adgst1AS1 gene, were obtained from fourth instar lan;ae of Anopheles dirus mosquito by reverse transcriptase PCR reactions. The nucleotide Sequences of these three cDNAs share > 67% identity and the translated amino acid Sequences share 61-64% identity. A comparison of the An. dirus to the An. gambiae enzymes shows that adGST1-2 versus agGST1-4. adGST1-3 versus agGST1-5 and adGST1-4 versus agCST1-3 have 85, 92 and 85% amino acid Sequence identity, respectively. which confirms that orthologous isoenzymes occur across anopheline species. These three proteins were expressed at high levels, approximately 15-20 mg from 200 mi of E. coli culture. The recombinant enzymes were purified by affinity chromatography on an S-hexylglutathione agarose column. The subunit sizes of adGST1-2. adGST1-3 and adGST1-4 are 24.3, 23.9 and 25.1 kDa. The recombinant enzymes have high activities with 1-chloro-2,3-dinitrobenzene (CDNB), detectable activity with 1,2-dichloro-4-nitrobenzene but markedly low activity with ethacrynic acid and p-nitrophenethyl bromide, adGST1-3 was shown to be the most active enzyme from the kinetic studies. Permethrin inhibition of CDNB activity, at varying concentrations of CDNB, was significantly different, being uncompetitive for adGST1-2, noncompetitive for adGST1-3 and competitive for adCST1-4. In contrast, permethrin inhibition with varying glutathione concentrations was noncompetitive for all three GSTs. Despite the enzymes being splicing products of the same gene and sharing identical Sequence in the N-terminal 35 amino acids, these GSTs show distinct substrate specificities-kinetic properties and inhibition properties modulated by the differences in the C-terminus. (C) 2001 Elsevier Science Ltd. All rights reserved.

Palavras-Chave: Glutathione S-Transferase; Anopheles Dirus; Mosquito; Isoenzymes; Alternatively Spliced Proteins

Imprenta: Insect Biochemistry and Molecular Biology, v. 31, n. 9, p. 867-875, 2001

Identificador do objeto digital: 10.1016/S0965-1748(01)00032-7

Descritores: Aedes aegypti - RNA

Data de publicação: 2001

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