Differential centrifugation, calcium precipitation, and ultrasonic disruption of midgut cells of Erinnyis ello caterpillars. Purification of cell microvilli and inferences concerning secretory mechanisms

Abstract

Subcellular fractions of the cells from the first and last third of midguts from Erinnyis ello caterpillars were obtained by conventional homogenization, followed by differential centrifugation or differential calcium precipitation, as well as by partial ultrasonic disruption. Aminopeptidase was enriched in the subcellular fractions, which in the electron microscope display mainly microvilli from the columnar cells (obtained by differential centrifugation and ultrasonic disruption), and also in the microvilli fraction obtained by differential precipitation. To account for the enzyme activities that sedimented with vesicles displaying brush borders, major amounts of the soluble glycosidases (cellobiase, N-acetylglucosaminidase, maltase, and trehalase) are assumed to be loosely bound to the cell glycocalyx, from where they are set free by homogenization and (or) freezing–thawing. Intracellular glycosidases seem to be bounded by membranes, which sediment together with vesicles that resemble secretory vesicles. The soluble form of amylase occurred mainly associated with the microvilli of anterior midgut cells and is supposed to be contained inside small vesicles, which are seen budding along columnar cell microvilli and fusing one with the 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.