Sequencing of Spodoptera frugiperda midgut trehalases and demonstration of secretion of soluble trehalase by midgut columnar cells
Contribuinte(s) |
UNIVERSIDADE DE SÃO PAULO |
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Data(s) |
20/10/2012
20/10/2012
2009
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Resumo |
Both soluble (SfTre1) and membrane-bound (SfTre2) trehalases occur along the midgut of Spodoptera frugiperda larvae. Released SfTre2 was purified as a 67 kDa protein. Its K(m) (1.6 mM) and thermal stability (half life 10 min at 62 degrees C) are different from the previously isolated soluble trehalase (K(m) = 0.47 mM; 100% stable at 62 degrees C). Two cDNAs coding for S. frugiperda trehalases have been cloned using primers based on consensus sequences of trehalases and having as templates a cDNA library prepared from total polyA-containing RNA extracted from midguts. One cDNA codes for a trehalase that has a predicted transmembrane sequence and was defined as SfTre2. The other, after being cloned and expressed, results in a recombinant trehalase with a K(m) value and thermal stability like those of native soluble trehalase. This enzyme was defined as SfTre1 and, after it was used to generate antibodies, it was immunolocalized at the secretory vesicles and at the glycocalyx of columnar cells. Escherichia coli trehalase 3D structure and sequence alignment with SfTre1 support a proposal regarding the residue modulating the pKa value of the proton donor. Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) FAPESP Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) CNPq |
Identificador |
INSECT MOLECULAR BIOLOGY, v.18, n.6, p.769-784, 2009 0962-1075 http://producao.usp.br/handle/BDPI/31003 10.1111/j.1365-2583.2009.00920.x |
Idioma(s) |
eng |
Publicador |
WILEY-BLACKWELL PUBLISHING, INC |
Relação |
Insect Molecular Biology |
Direitos |
restrictedAccess Copyright WILEY-BLACKWELL PUBLISHING, INC |
Palavras-Chave | #trehalase #secretory mechanism #recombinant trehalase #catalytic groups #trehalase membrane release #PERITROPHIC MEMBRANE #DIGESTIVE ENZYMES #LARVAL MIDGUT #MICROVILLAR MEMBRANES #POLYACRYLAMIDE GELS #BICINCHONINIC ACID #MOLECULAR-CLONING #ERINNYIS-ELLO #ACTIVE-SITE #BOMBYX-MORI #Biochemistry & Molecular Biology #Entomology |
Tipo |
article original article publishedVersion |