Chemical determinations in microvillar membranes purified from brush-borders isolated from the larval midgut of one coleoptera and two diptera species

Abstract

Brush-borders (microvilli) were isolated from different midgut regions of larvae of Tenebrio molitor (Coleoptera), Rhynchosciara americana (lower Diptera), and Musca domestica (higher Diptera) by differential precipitation from homogenates prepared as previously described. The microvillar preparations were treated with the chaotropic salts lithium diiodosalicylate and sodium thiocyanate in order to disrupt microvilli into microvillar membranes and core (cytoskeleton) material. Marker enzymes were extensively inactivated and there was not a selective release of cytoskeleton elements from the microvillar membranes. Insect microvillar preparations were also treated with hyperosmotic Tris buffer, then diluted, centrifuged, and the purified microvillar membranes were recovered from the resulting pellet. Specific activities of marker enzymes in purified membranes were 1.5-3.0-fold higher than in the original microvillar preparations with a final yield of about 20%. Contamination by soluble proteins was under 0.3% and by other membranes never exceeded 5%, as judged by chromatography in Sepharose 4B and sucrose gradient ultracentrifugation. In comparison with mammals, insect membranes are rich in carbohydrates (all insects studied), cholesterol (T. molitor), lipids (T. molitor), and protein (M. domestica). The densities, and the ratio of lipid to protein in the microvillar membranes of T. molitor is lower than that in the two species of Diptera. This agrees with the fact that microvillar hydrolases are more important in Diptera than in Coleoptera digestion.