Nature of the anchors of membrane-bound aminopeptidase, amylase, and trypsin and secretory mechanisms in Spodoptera frugiperda (Lepidoptera) midgut cells.

Spodoptera frugiperda larvae have a microvillar aminopeptidase and both soluble and membrane-bound forms of amylase and trypsin. Membrane-bound aminopeptidase is solubilized by glycosyl phosphatidylinositol-specific phospholipase C (GPI-PLC) and detergents, suggesting it has a GPI anchor. Membrane-bound trypsin is not affected by GPI-PLC, although it is solubilized by papain and by different detergents. Membrane-bound amylase is similar to trypsin, although once solubilized in detergent it behaves as a hydrophilic protein. Musca domestica trypsin antiserum cross-reacts with only one polypeptide from S. frugiperda midgut. With this antiserum, trypsin was immunolocalized in the anterior midgut cells at the microvillar surface and on the membranes of secretory vesicles found in the apical cytoplasm and inside the microvilli. The data suggest that in this region trypsin is bound to the secretory vesicle membrane by a hydrophobic anchor. Vesicles migrate through the microvilli and are discharged into the lumen by a pinching-off process. Trypsin is then partly processed to a soluble form and partly, still bound to vesicle membranes, incorporated into the peritrophic membrane. In posterior midgut cells, trypsin immunolabelling is randomly distributed inside the secretory vesicles and at the microvilli surface, suggesting exocytosis. Amylase probably follows a route similar to that described for trypsin in anterior midgut, although membrane-bound forms (peptide anchor) solubilize apparently as a consequence of a pH increase inside the vesicles.

An immunocytochemical investigation of trypsin secretion in the midgut of the stablefly, Stomoxys calcitrans.

Musca domestica trypsin antibody cross-reacts with polypeptide bands of M(r) 25,000 and 30,000 showing proteolytic activity from Stomoxys calcitrans midgut extracts. Secretory granules from the main enzyme-secreting region, the opaque zone, stained heavily with the trypsin antibody in both unfed and blood-fed flies. Heterogeneous staining of granules suggests the unequal distribution of trypsin in secretory granules. This is also consistent with the occurrence of non-parallel secretion, which is also suggested by the possible preferential release of smaller, heavily stained secretory granules in fed flies. The predigestive, anterior midgut region responsible for rapid dehydration of the blood meal, the reservoir zone, contains a different population of secretory granules which stain heavily with trypsin antibody. This zone contains 20% of the midgut trypsin activity in unfed flies; trypsins are held here as proenzymes which are probably only activated postsecretion. In the midgut lumen of both unfed and blood-fed flies, trypsin is mainly immunolocalized in the ectoperitrophic space. Enzyme assays suggest that 5-15% of the lumenal trypsin is associated with the peritrophic matrix. The finding of intact secretory granules plus cell debris in the ectoperitrophic space of opaque and lipoid zones of blood-fed flies supports the contention that some trypsin is released by apocrine secretion in this insect.

Regional distribution and substrate specificity of digestive enzymes involved in terminal digestion in Musca domestica hind-midguts.

One membrane-bound alpha-glucosidase and two soluble alpha-glucosidases were isolated from homogenates of the hind-midgut, the main digestive region in Musca domestica larvae. The membrane-bound alpha-glucosidase and the low-Mr soluble alpha-glucosidase hydrolyze maltopentaose better than maltose, maltotriose, and maltotetraose, the reverse being true for the high-Mr soluble alpha-glucosidase. A membrane-bound glucoamylase previously described in Musca domestica midgut was shown by gradient centrifugation and dialysis against EDTA to result from the combined action of an amylase and an alpha-glucosidase. The determination of amylase, alpha-glucosidases, soluble and membrane-bound carboxypeptidase A, membrane-bound aminopeptidase and dipeptidase along the tissue and luminal contents of the hind-midgut is described. The data support a proposal concerned with how starch and protein are digested in Musca domestica larval hind-midguts and where and how midgut glycosidases and peptidases are secreted.