The studies described in this application are designed to continue to elucidate the early events involved in the biosynthesis, processing and intracellular transport of intestinal glycoprotein enzymes destined for insertion into the microvillous membrane, and the factors controlling the ontogeny of these mechanisms. Two enzymes will be studied in detail and compared: sucrase-isomaltase (E.C. - E.C. and lactase-phlorizin hydrolase (E.C. - E.C. Using immunochemical and cell fractionation techniques, the labeling patterns of newly synthesized enzymes will be examined in subcellular fractions (microsomes, Golgi, microvillous membranes) of rat intestine at various times during development and maturation. The subunit composition and assembly of potential microsomal biosynthetic precursors will be identified and characterized using two dimensional polyacrylamide gel electrophoresis and fluorography. Homologies in primary structure between microsomal and microvillous forms of these enzymes will be defined by cyanogen bromide cleavage; core glycosylation will be studied by direct labeling experiments in the absence and presence of tunicamycin, and treatment of microsomal precursors with endo-Beta-N-acetylglucosaminidase-H, followed by analysis of sugars released. Analysis of the role of sugar-lipid intermediates in the early glycosylation of sucrase-isomaltase and lactase-phlorizin hydrolase will be undertaken, and thr role of dolichol in this process investigated. Dolichol content of intestinal tissue will be quantified by high performance liquid chromatography, and its synthesis delineated using labelled acetate and mevalonate as precursors. The role of dolichol-linked glycosylation of membrane proteins during fetal and post-natal development will also be analyzed, and if possible, the effects of compactin on this process elucidated. In addition, the impact of cholesterol and sugar feeding on dolichol synthesis will be studied. Detailed analysis of the early intracellular steps governing the biosynthesis of these enzymes should allow understanding not only of their developmental control but also the mechanisms responsible for their transport to the intestinal cell surface. In addition, elucidation of mechanisms controlling the post-weaning decline in lactase activity may yield insights into human adult-onset lactose intolerance.

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Tufts University
United States
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Mobassaleh, M; Donohue-Rolfe, A; Jacewicz, M et al. (1988) Pathogenesis of shigella diarrhea: evidence for a developmentally regulated glycolipid receptor for shigella toxin involved in the fluid secretory response of rabbit small intestine. J Infect Dis 157:1023-31
Biller, J A; Montgomery, R K; Grand, R J et al. (1987) Characterisation of humoral trophic factors involved in intestinal adaptation using a 3T3 cell growth factor assay. Gut 28 Suppl:243-7
Buller, H A; Montgomery, R K; Sasak, W V et al. (1987) Biosynthesis, glycosylation, and intracellular transport of intestinal lactase-phlorizin hydrolase in rat. J Biol Chem 262:17206-11
Biller, J A; Montgomery, R K; Grand, R J et al. (1986) Use of a 3T3 cell growth factor assay for the delineation and characterization of humoral trophic factors involved in intestinal adaptation in the rat. Gastroenterology 91:448-55
Fuchs, G; Mobassaleh, M; Donohue-Rolfe, A et al. (1986) Pathogenesis of Shigella diarrhea: rabbit intestinal cell microvillus membrane binding site for Shigella toxin. Infect Immun 53:372-7
Jonas, M M; Montgomery, R K; Grand, R J (1985) Intestinal lactase synthesis during postnatal development in the rat. Pediatr Res 19:956-62
Mobassaleh, M; Montgomery, R K; Biller, J A et al. (1985) Development of carbohydrate absorption in the fetus and neonate. Pediatrics 75:160-6
Grand, R J; Montgomery, R K; Perez, A (1985) Synthesis and intracellular processing of sucrase-isomaltase in rat jejunum. Gastroenterology 88:531-8