α-Mannosidases intestinal from Tenebrio molitor (Coleoptera) larvae / α-Manosidases intestinais da larva de Tenebrio molitor (Coleoptera)

AUTOR(ES)
DATA DE PUBLICAÇÃO

2008

RESUMO

Studies of intestinal function were prompted after noticing that the gut is a huge and relatively unprotected interface between the insect and the environment and can thus be used as a target for pest control. In this context, our work involves the purification and characterization of an soluble alpha-mannosidase and detection of a membrane α-mannosidase. α-Mannosidases are a family of exoglycosidases which hydrolyse α-D-mannosyl residues from terminal non-reducing end of oligossacharides. These enzymes are implicated in the catabolism of carbohydrates and N-linked protein glycosylations in insects, but little is known on this biochemistry. T.molitor is a Coleoptera studied in our laboratory because of its relevance as agricultural pest and its position at a strategic point in the phylogenetic tree of insects. α-Mannosidase is more active in the anterior and middle midgut content of T.molitor larvae, although there is a significant activity in the membrane fraction. To confirm the existence of this membrane enzyme, microvilli were purified by differential precipitation with calcium. Aminopeptidase was used as a marker, since it is known that it is a typical microvilar membrane enzyme. Most α-mannosidase activity is soluble. This led us to purify this enzyme for further characterization. The purification of T. molitor α-mannosidase was attained by using a combination of four chromatographic steps: an anion-exchange chromatography in Hitrap Q XL (Amersham/Bioscience), two gel filtration chromatographies, one in Superdex 200 and another in Superdex 75 (Amersham/Bioscience) using an AKTA system, and the last step is a Hydrophobic cromatography in Phenyl Superose. Two peaks of activity were resolved: Man 1 and Man 2, suggesting the existence of two soluble α-mannosidases, differing only in hydrophobicity. The optimum pH of the α- mannosidases is 5.6 and the molecular mass is 123 KDa determined by gel filtration and 70 KDa in the case of SDS PAGE. This suggests that the holoenzyme has two subunits. In a native gel revealed with the fluorescent substrate (methylumbelliferyl-α-D-mannopyranoside) only one band of activity is seen. Man 2 has pI 3.38. T. molitor α-mannosidases followed Michaelis-Menten kinetics with a Km value of 0.84 mM for Man 1 and 0.62 mM for Man 2 using p-nitrophenyl-α-D-mannopyranoside as substrate. Inhibition tests were made with typical inhibitors of α-mannosidases: one is the 1-deoxymannojirimycin and the other is the Swainsonine. The Ki for the first was of 0.12 mM for Man 1 and 0.15 mM for Man 2 and for the second was 67.8 nM for Man 1 and 63 nM for Man 2. Both were competitive inhibitors. The fact that the enzymes are inhibited only by swainsonine in reasonable concentrations, allows us to classify them as type II. This suggests that they are derived from the lysosomal form, although they have an altered optimum pH.

ASSUNTO(S)

coleoptera bioquímica animal enzimas enzymes microvilli tenebrio molitor α-mannosidase caracterização cinética digestão animal digestão kinetic characterization insects (metabolism) digestion α-manosidase microvilosidades tenebrio molitor insetos (metabolismo) coleoptera

ACESSO AO ARTIGO

Documentos Relacionados