Immunocytochemical localization of a brush border hydrolase, lactase, in newborn suckling rat jejunum

Abstract

Lactase-phlorizin hydrolase (LPH, EC 3.2.1.23), an integral membrane glycoprotein of the small intestinal brush border, converts lactose, the main carbohydrate in milk, to its monosaccharide components. Although the activity of LPH is high in suckling rats, little is known about its distribution within the intracellular compartments of the secretory pathway and brush border. We present the first description of the ultrastructural localization of LPH in the neonatal jejunum of suckling rat pups.Pieces of jejunum from 12- to 14-d-old suckling rat pups from three litters of Sprague- Dawley rats were fixed with 4% freshly prepared paraformaldehyde in 100 mM phosphate buffer, pH=7.40, for 4 h, and stored in 1% paraformaldehyde, at 4°C, until further processing. The samples were sectioned after cryoprotection in 2.3 M sucrose in an RMC MT-7 ultramicrotome equipped with CR21 cryoattachment. Ultrathin cryosections were collected on Formvar-coated, carbon-evaporated nickel grids. The nonspecific binding sites were blocked by 1% heat-inactivated newborn calf serum in 10 mM Trizma buffer, pH=7.60, containing 500 mM NaCl, 0.05% NaN3and 20 mM glycine (buffer A).