Carbohydrate digestion at the small intestinal surface depends upon maintenance of appropriate levels of constituent oligosaccharides at the brush border membrane and information on the intracellular synthesis, assembly and transfer of these glycoprotein hydrolases in intact mammalian intestine under physiological conditions is beginning to emerge. The rat model will be used to examine the mechanism of induction of sucrase-Alpha-dextrinase synthesis at the molecular level. Paired intestinal segments from animals fed a carbohydrate-free diet for 7 days will be perfused with either sucrose plus saline or saline alone and then given a 5 minute intraintestinal pulse of 35S-methionine followed by a non-radioactive methionine chase of 15-180 minutes. Subcellular organelles (Er, Golgi, laterobasal membrane, brush border membrane) will be isolated and their proteins solubilized and sucrase-Alpha-dextrinase specifically immunoprecipitated. Newly synthesized sucrase-dextrinase will be quantified by scintillation counting and the molecular species analyzed by SDS-electrophoresis-autoradiography. In addition to sucrose, glucose plus fructose, lactulose and acarbose, a sucrase inhibitor, will be studied as putative inducers of de novo synthesis; the role of insulin, VIP, glucagon and secretin given intravenously with or without glucose will also be assessed. After the qualitative and quantitative effects on induction of synthesis, assembly and membrane insertion have been established, mRNA specific for sucrase-Alpha-dextrinase will be isolated from polysomes whose sucrase-dextrinase (S-D) nascent chains bind specifically to anti-S-D. The S-D mRNA will be translated in the rabbit reticulocyte system and will be used as a template for 32P-labeled S-D cDNA in the presence of reverse transcriptase. The 32P-cDNA probe will then be used in hybridization assays to estimate both mRNA and specific S-D DNA in normal rats and those whose sucrase-dextrinase has been induced by carbohydrate. The regulation of sucrase-dextrinase assembly will also be examined in the HT29 and CACO-2 colon cancer cell lines that synthesize sucrase-dextrinase under conditions favoring differentiation. Experiments analogous to those in the intact rat will be carried out, and, in particular, the role of glycosylation in the ER and Golgi on synthesis and assembly will be examined by the use of inhibitors of glycosylation such as tunicamycin, castanospermine and swainsonine. In the aggregate, these experiments should provide new information at the molecular level on the regulation of sucrase-dextrinase assembly by luminal carbohydrate.
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