Microvillar and secreted digestive enzymes from Musca domestica larvae. Subcellular fractionation of midgut cells with electron microscopy monitoring

Abstract

Subcellular fractions of the cells of fore-midgut and hind-midgut of Musca domestica larvae were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation. Aminopeptidase follows a distribution pattern among fractions similar to that of γ-glutamyl transferase (a plasma membrane marker) regardless of the method of fractionation used. Furthermore, the aminopeptidase activity present in a sediment obtained by centrifugation of midgut tissue homogenates at 600 g for 10 min accompanies that of γ-glutamyl transferase during sucrose gradient ultracentrifugation of the sediment (apparent buoyant density: 1.16 g/cm 3). Maltase and trehalase activity follows approximately that of aminopeptidase, except for some soluble activity. Amylase and lysozyme are soluble enzymes found mainly in membrane-bound vesicles (apparent buoyant density: 1.21 g/cm 3) which sediment at 25,000 g for 10 min, and from which they are set free on freezing and thawing. The data favor the view that aminopeptidase and the majority of the maltase and trehalase are bound to the plasma membranes covering the microvilli of the columnar cells, whereas amylase and lysozyme are soluble enzymes which are secreted into the midgut lumen by exocytosis. Trypsin secretion is proposed to occur mainly through the extrusion of vesicles from the sides of hind-midgut cell microvilli.