Fisiologia e bioquímica da digestão em Erinnyis ello (Lepidoptera Sphingidae)

Abstract

E/ilnny<Í6 dllo midgut is composed of goblet and columnar cells. Goblet cells have a cavity, which is formed by invagination of the apical membrane. The infolded apical membrane shows modified microvilli, which sometimes (posterior midgut) or always (anterior and middle midgut) contain mitochondria. The cytoplasmic side of the membrane of the microvilli that contain mitochondria are studded with small particles, which are supposed to be a K -ATPase responsible for K extrusion. Columnar cells display microvilli with vesicles pinching off from their tips (anterior and middle midgut) or with a large number of double membrane spheres budding along their length (posterior midgut). Basal infoldings (with associated mitochondria) in columnar cells occur in a parallel açsay with many openings to the underlying space (posterior midgut) or are less organized with few openings (anterior and middle midgut). The results suggest that the anterior and middle region of the midgut absorb water, whereas the posterior region secreteS it. Supports this belief the observations carried out after introduction of amaranth into the oesophagus of E. alto larvae and injection of amaranth into E. alio haemocoele. Some physical and kinetical properties of E. alio digestive enzymes were determined. Amylase and trypsin are found in a soluble and in a membrane-bound form. Soluble amylase (Mr 48000; pHQ 9.8; Km for starch 0.12%, pi 6.0) and soluble trypsin (Mr 55000; pHo9.5; Km for BAPA 0.17 mM; pi 4.6) are supposed to be derived frcm membrane bound forms. Aminopeptidase (pH0 8,0; Km for LPNA 0,36 mM) occurs only membrane-bound, whereas carboxypeptidase is found in a soluble (a hexamer composed of three different Mr 17000 - subunits; pH0 9.0; Km for ZGlyPhe 0.18 mM) and in a membrane-bound (Mr 136000; pHo 9.0) form. 3-N-Acetylglucosaminidase (Mr 142000; pHQ 4.5; Km for pNPNAG 0.027 mM), 3-fructosidase (Mr 80000; pH 6.0; km for raffinose and sucrose 30 mM) , a-galactosidase (a dimer composed of two identical Mr 39000 - subunits; pH 6.0; Km for pNPaGal 7.4 mM), maltase (a tetramer of identical Mr 34000 - subunits; pHo 5.8; Km for maltose 1.4 mM, for pNPaGlu 0.63 mM; Ki por Tris 0.34 mM) and trehalase (Mr 103000; pHo 5.7; Km 0.69 mM) are mainly soluble. A more ccmprehensive study was accomplished with cellobiase. This enzyme is soluble and has pHo 6.5, Mr 130000 and pi 6.8. The cellobiase hydrolyses 3-Dglucosides, 3-D-galactosides, 3-D-fucosides and 3-D-xylosides at the same active site. It exhibits rapid-equilibrium kinetics. The hydrolysis of the 3-D-glucosidic bond catalyzed by the 3-D-glucosidase occurs without inversion of configuration, through a mechanism similar to acid catalysis which involves the intermediary formation of a carbonium ion. In an attempt to study the spatial organization of the digestive process in E. alio larvae, several enzymes were assayed at different sites in E. alio midguts. The enzymes responsible for the initial attack to the food (amylase and trypsin) penetrate into the endoperitrophic space, whereas the others (3-N-acetylglucosaninidase, aminopeptidase, carboxypeptidase, 3-fructosidase, a-galactosidase, maltase and trehalase) are absent from there. The subcellular distribution of enzymes in cells from the anterior and posterior midgut were studied by the differential centrifugation of tissue homogenates and by the purification of cell microvilli using sound disruption or calcium differential preciptation tecniques. The results showed that the aminopeptidase is bound to the plasma membrane covering the columnar cells microvilli, whereas cellobiase, 3-fructosidase, a-galactosidase, maltase and trehalase are soluble enzymes, which seem to be secreted by exocytosis and then becoming trapped into the glycocalyx, mainly of the posterior midgut. The membrane-associated forms of amylase and trypsin seem to be true integral proteins displaying intermolecular interactions with cytoskeletal elements. The soluble intracellular forms of those enzymes are supposed to be contained inside small vesicles, which are seem budding along columnar cells microvilli and fusing one with each other and with the tips of the microvilli from the anterior midgut cells. Secretory mechanisms are discussed in the light of the evidence that the posterior midgut secretes, whereas the anterior midgut absorbs water. The low excretion rates of amylase and trypsin, the major activities that those enzymes display in the anterior midgut contents and the above mentioned ultrastructural data led to the proposal of a mechanism by which the enzymes are recovered from the undigested food before they are excreted.